BR112016017388B1 - POLYPEPTIDE WITH XYLANASE ACTIVITY AND ITS PRODUCTION METHOD, FEED OR FOOD FOR ANIMALS AND METHOD FOR SOLUBILIZING ARABINOXYLAN IN A - Google Patents

Abstract

PROTEIN. The present invention relates to an isolated polypeptide having xylanase activity, selected from the group consisting of: a) a polypeptide comprising an amino acid sequence that has at least 87% identity with SEQ ID NO: 1; b) a polypeptide encoded by a polynucleotide that has at least 87% identity with SEQ ID NO: 2 or c) a fragment of a polypeptide from a) or b) which fragment has xylanase activity.

Description

FIELD OF THE INVENTION

[001] The present invention relates to innovative synthetic xylanases and the use of said xylanases in applications, including animal feed, in the fermented beverage and malting industry, in the treatment of raw materials containing arabinoxylan such as arabinoxylan-based materials grains, for example, in the production of biofuel or other fermentation products, including biochemicals (for example, bioisoprene), and/or in the gluten-wheat starch separation industry, and to methods using these xylanases, and also to compositions (such as feed additive compositions) comprising said xylanases.

BACKGROUND OF THE INVENTION

[002] For many years, endo-β-1,4-xylanases (EC 3.2.1.8) (referred to in the present invention as xylanases) were used for the modification of complex carbohydrates derived from plant cell wall material. It is well known in the art that the functionality of different xylanases (derived from different microorganisms or plants) differs enormously. Xylanase is the name given to a class of enzymes that degrade the linear polysaccharide beta-1,4-xylan into xylo-oligosaccharides or xylose, thus breaking down hemicellulose, one of the main components of the plant cell wall.

[003] Based on structural and genetic information, xylanases have been classified into different glycoside hydrolase (GH) families (Henrissat, (1991) Biochem. J. 280, 309-316).

[004] Initially, all known and characterized xylanases belonged to the GH10 or GH11 families. Additional studies then identified several other types of xylanases belonging to the GH5, GH7, GH8 and GH43 families (Collins et al. (2005) FEMS Microbiol Rev., 29(1), 3-23).

[005] So far, the GH11 family differs from all other GHs, being the only family consisting only of xylan-specific xylanases. The structure of GH11 xylanases can be described as a β-swirl structure or a folded structure as an all β-strand sandwich ( Himmel et al. 1997 Appl. Biochem. Biotechnol. 63-65, 315-325 ). GH11 enzymes have a catalytic domain of about 20 kDa.

[006] The GH10 xylanases have a catalytic domain with molecular weights in the range of 32 to 39 kDa. The structure of the catalytic domain of GH10 xylanases consists of an eightfold β/α barrel (Harris et al. 1996 - Acta. Crystallog. Sec. D 52, 393-401).

[007] Three-dimensional structures are available for a large number of enzymes of the GH10 family, and the first to be solved were those of xylanase A from Streptomyces lividans (Derewenda et al. J Biol Chem 1994 Aug 19; 269(33) 20811-4) , Cex endoglucanase from C. fimi (White et al. Biochemistry 1994, Oct 25; 33(42) 12546-52) and Xyn10A from Cellvibrio japonicus (formerly known as Pseudomonas fluorescens subsp. xylanase A) (Harris et al. Structure 1994 Nov 15; 2(11) 1107-16.). As members of the GHA clan, they have a classic barrel fold (α/β)8 TIM with the two key active site glutamic acids located at the C-terminal ends of the beta 4 (acid/base) and 7 (nucleophile) strands ( Henrissat et al Proc Natl Acad Sci U S A 1995 Jul 18;92(15) 7090-4).

[008] Comprehensive studies to characterize the functionality of xylanases have been carried out on pure and well-characterized substrates (Kormelink et al., 1992 Characterization and mode of action of xylanases and some accessory enzymes. Doctoral Thesis, Agricultural University Wageningen, Netherlands (175 pages , abstracts in English and Dutch)). These studies show that different xylanases have different specific requirements with regard to replacement of the xylose backbone of arabinoxylan (AX). Some xylanases require three unsubstituted xylose residues to hydrolyze the xylose backbone, others require only one or two. The reasons for these differences in specificity are believed to be caused by the three-dimensional structure within the catalytic domains which, in turn, depends on the primary structure of the xylanase, i.e., the amino acid sequence. However, the translation of these amino acid sequence differences into differences in xylanase functionality has so far not been documented when the xylanase acts in a complex environment, such as a material of plant origin, for example, in animal feed.

[009] The xylanase substrates in the material of plant origin, for example, in wheat, have traditionally been divided into two fractions: The non-water extractable AX fraction (WU-AX) and the water extractable AX (WE-AX). There have been several explanations as to why there are two different fractions of AX. The older literature (D'Appolonia and MacArthur - (1976, Cereal Chem. 53. 711-718) and Montgomery and Smith (1955, J. Am. Soc. 77. 3325-332) describe very large differences in the degree of substitution between WE-AX and WU-AX. The highest degree of substitution was found in WE-AX. This was used to explain why some of the AX were extractable. The high degree of substitution made the polymer soluble, compared to a lower degree of substitution, which could form hydrogen bonds between the polymers and, consequently, precipitation.

[0010] It was believed that the difference between the functionality of the different xylanases was due to differences in the specificity of the xylanase and thus its preference for WU-AX or WE-AX substrates.

[0011] Xylanase enzymes have been reported from nearly 100 different organisms, including plants, fungi, and bacteria. Xylanase enzymes are classified into several of over 40 families of glycosyl hydrolase enzymes. Glycosyl hydrolase enzymes, which include xylanases, mannanases, amylases, β-glucanases, cellulases and other carbohydrases, are classified based on properties such as the sequence of amino acids, their three-dimensional structure and the geometry of their catalytic site (Gilkes, et al. ., 1991, Microbiol. Reviews 55: 303-315).

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 shows the mature polypeptide of a synthetic xylanase called, in the present invention, SynXyn92 (SEQ ID NO: 1).

[0013] Figure 2 shows the polynucleotide sequence of a synthetic xylanase called, in the present invention, SynXyn92 (SEQ ID NO: 2).

[0014] Figure 3 shows the mature polypeptide of a synthetic xylanase called, in the present invention, SynXyn85 (SEQ ID NO: 3).

[0015] Figure 4 shows the polynucleotide sequence of a synthetic xylanase called, in the present invention, SynXyn85 (SEQ ID NO: 4).

[0016] Figure 5 shows the mature polypeptide of a synthetic xylanase called, in the present invention, SynXyn89 (SEQ ID NO: 5).

[0017] Figure 6 shows the polynucleotide sequence of a synthetic xylanase called, in the present invention, SynXyn89 (SEQ ID NO: 6).

[0018] Figure 7 shows the mature polypeptide of a synthetic xylanase called, in the present invention, SynXyn72 (SEQ ID NO: 7).

[0019] Figure 8 shows the polynucleotide sequence of a synthetic xylanase called, in the present invention, SynXyn72 (SEQ ID NO: 8).

[0020] Figure 9 shows the mature polypeptide of a synthetic xylanase called, in the present invention, SynXyn80 (SEQ ID NO: 9).

[0021] Figure 10 shows the polynucleotide sequence of a synthetic xylanase called, in the present invention, SynXyn80 (SEQ ID NO: 9).

[0022] Figure 11 shows the mature polypeptide of a synthetic xylanase called, in the present invention, SynXyn93 (SEQ ID NO: 11).

[0023] Figure 12 shows the polynucleotide sequence of a synthetic xylanase called, in the present invention, SynXyn93 (SEQ ID NO: 12).

[0024] Figure 13 shows a schematic representation of the expression vector for the synthetic xylanases (pTTTpyr2-synXyn_VAR).

[0025] Figure 14 shows an alignment of the mature polypeptide sequences of the synthetic xylanases - the CLUSTAL Omega multiple sequence alignment was used with the default parameter setting (Matrix: Gonnet; Gap Penalty: 10; Gap Length: 0, two).

[0026] Figure 15 shows the results of a pellet test on the processing stability of the synthetic xylanase SynXyn92 - at processing temperatures of 90°C.

[0027] Figure 16 shows the reduction of viscosity in grain-based material by the synthetic enzyme SynXyn93 according to the present invention, compared to a positive control enzyme. These data show that the SynXyn93 enzyme has no activity during the pre-treatment step (60°C), but recovers in the liquefaction step (85°C). The viscosity reduction continues during the liquefaction step at 85°C, indicating that SynXyn93 has significant activity at this elevated temperature. Final viscosities with SynXyn93 are 52% to 55% lower than the blank.

[0028] Figure 17 shows the reduction of viscosity in grain-based material by the synthetic enzyme SynXyn93 according to the present invention, compared to a positive control enzyme. These data confirm the increased thermostability of SynXyn93 compared to the positive control enzyme. SynXyn93 shows a viscosity reduction of 54% to 60% compared to the blank, while for the positive control enzyme the reduction was only 40% to 41%. The final viscosity with SynXyn93 is lower than with the positive control enzyme.

SUMMARY OF THE INVENTION

[0029] A seminal discovery of the present invention is that synthetic xylanases can be designed that, in addition to having the ability to decompose (solubilize) insoluble arabinoxylans (AXinsol), also have other properties that make them especially useful in applications, such as feed for animals, in the fermented beverage and malting industry, in the treatment of raw materials containing arabinoxylan such as grain-based materials, e.g. in the production of biofuel or other fermentation products, including biochemical compounds (e.g. bioisoprene) , and/or in the gluten-wheat starch separation industry. For example, the synthetic xylanases demonstrated here have surprising thermostability, have surprisingly high recovery, eg residual activity after heat treatment (e.g., during the pelletizing process) and are pepsin resistant.

[0030] During the pelleting process, the enzyme (eg a feed containing the enzyme) is conditioned for 30 seconds at 90°C.

[0031] During the pelletizing process, an enzyme can be formulated into a substrate, for example, wheat, and can be formulated into a premix, for example, a corn/soy mixture (such as 61.1% corn , 31.52% Hipro Soya 48 Soybean, 4.00% Soybean Oil, 0.40% Sodium Bicarbonate, 0.25% Leghennen Vitamins/Minerals, 0.20% DL Methionine, 1.46 % dicalcium phosphate, 1.16% limestone).

[0032] Xylanase can be included at a level that will ensure that a final target dosage of, for example, 20,000 XU/kg feed is achieved. The premix can be prepared by mixing the formulated enzyme(s) into the substrate, e.g. wheat, forming a feed mix, e.g. 10 kg corn/soy feed mix, and mixing for a specified time, for example 10 minutes.

[0033] The premix can be added to the feed, for example, 110 kg of feed, and mixed for a specific time, for example, 10 minutes, before conditioning. The feed comprising the enzyme is typically conditioned for 30 seconds at 90°C prior to pelleting.

[0034] The feed comprising the enzyme can be treated with dry steam to reach a target temperature of 90°C after 30 seconds.

[0035] The term "conditioning" or conditioning" as used herein means mixing the feed/enzyme mixture and treating it with dry steam to reach a target temperature of 90°C after 30 seconds.

[0036] After conditioning, the feed/enzyme mixture can be formatted as pellets. Formatting of the pellets can be done by any of the conventional means known to the person skilled in the art. Pellets can be shaped by the pelletizing process described herein.

[0037] For the first time the present inventors were able to fully express synthetic polypeptides having xylanase activity and enhanced properties.

[0038] The synthetic xylanases taught in the present invention are GH10 xylanases.

[0039] In particular, the synthetic xylanases of the present invention effectively decompose (solubilize) AXinsol from a wide range of substrates, including corn, wheat, DDGS, etc., in particular, corn and corn-based substrates, in particular , wheat (including wheat-based) and corn (including corn-based) products. This is in contrast to previously known enzymes, which are often inferior in solubilizing AXinsol on maize or maize based substrates or which are not efficient on both wheat and maize based substrates.

[0040] Furthermore, the synthetic xylanases of the present invention are particularly suitable not only for decomposing (solubilizing) AXinsol, but also for effectively decomposing (or degrading) the solubilized polymers. By being able to effectively decompose (degrade) (quickly) solubilized polymers (obtained by dissolving AXinsol), a (rapid) reduction in viscosity is obtained or solubilized polymers (obtained by dissolving AXinsol) cannot contribute to the increase in viscosity. This last effect is essential in some of the claimed applications.

[0041] Without wishing to be bound by theory, the synthetic enzyme of the present invention primarily releases polymers, which do not contribute to viscosity due to the fact that the released polymers are short.

[0042] Typically, conventional xylanases can decompose AXinsol, but often lead to an increase in the viscosity of the mixture. This higher viscosity is disadvantageous in many applications.

[0043] Without wishing to be bound by theory, although some conventional xylanases decompose AXinsol, they lead to an increase in high molecular weight soluble degradation products, which leads to an increase in viscosity in the mixture.

[0044] Additionally or alternatively, and again without wishing to be bound by theory, conventional xylanase enzymes can decompose AXinsol, but as they do not degrade the high molecular weight solubilised products fast enough, the viscosity in the mixture is not ideal. In contrast, with the methods and uses of the present invention, synthetic xylanases decompose AXinsol without increasing viscosity and/or reduce viscosity rapidly compared to conventional enzymes. Without being bound by theory, it is believed that high molecular weight products are not formed by the enzymes of the present invention.

[0045] It has been observed that the enzymes of the present invention, and as described herein, not only decompose (solubilize) insoluble arabinoxylans (AXinsol) from a wide range of substrates, including corn, wheat, DDGS, etc., in particular, corn and corn-based substrates, in particular wheat (including wheat-based) and corn (including corn-based) products, but also effectively ensure that the viscosity is not high and/or reduce the viscosity . Without being bound by theory, it is believed that high molecular weight products are not formed by the enzymes of the present invention.

[0046] Thus, the present invention relates to enzymes capable of solubilizing pentosans, in particular, xylan-containing materials such as arabinoxylans, in particular, insoluble arabinoxylans. In particular, the enzyme is particularly suitable for the solubilization of pentosans, in particular xylan-containing materials such as arabinoxylans, in particular insoluble arabinoxylans, on a broad spectrum of substrates, including corn-based substrates.

[0047] Many of the xylanases marketed for use in animal feeds to solubilize pentosans are GH11 enzymes. It was considered by those skilled in the art that GH10 xylanases were not as strong in solubilizing pentosans, particularly AXinsol, compared to GH11 xylanases. Surprisingly, the synthetic xylanases disclosed here, which are GH10 xylanases, have been found to be particularly suited to degrading AXinsol on a broad spectrum of substrates, including corn-based substrates. Surprisingly, the present inventors have observed that the synthetic GH10 xylanases of the present invention outperform commercial GH11 xylanases in their ability to solubilize pentosans. Furthermore, synthetic GH10 xylanases are thermostable.

[0048] The fact that the present enzymes effectively degrade AXinsol from corn and corn-based substrates is significantly advantageous as corn contains much more AX in insoluble form compared to other cereals such as wheat and rye , for example. Therefore, only xylanases that can decompose AXinsol can show significant benefit for animals fed a corn-based diet, such as a soy-corn diet, for example.

[0049] It was completely unexpected that a GH10 xylanase would degrade AXinsol so well in cereals, particularly corn or corn-based substrates.

[0050] The enzymes of the present invention are capable of effectively (and rapidly) degrading the polymers and oligomers that are produced by the degradation of AXinsol or that are present in the grain-based material. This leads to an unexpected advantage for the synthetic xylanases taught here because they are particularly suited to various applications to maintain low viscosity or to reduce viscosity, for example in animal feeds; in the production of beer and/or malt; in the production of grain-based glucose, eg for further processing of biofuels and/or biochemicals (eg bioisoprene); or in the gluten-wheat starch separation industry for the production of starch, for example.

[0051] Especially, it was found that the degradation product on average is shorter for the GH10 enzymes tested here compared to the GH11 enzymes. This means that degradation products do not contribute or cause an increase in viscosity.

[0052] Based on these findings, the synthetic xylanases according to the present invention can be used to degrade a xylan-containing material, particularly arabinoxylans, particularly AXinsol. Additionally or alternatively, xylanases according to the present invention can be used to degrade soluble polymers (e.g. oligomers) which are produced from degradation of AXinsol or which are present (naturally) in grain-based materials. Surprisingly, it has been found that variant xylanases according to the present invention can be used to degrade a xylan-containing material, particularly arabinoxylans, particularly AXinsol, and to degrade soluble polymers (e.g. oligomers) that are produced from from the degradation of AXinsol.

[0053] Such enzymes find useful application in many industries, including feed, brewing or malting, in the treatment of raw materials containing arabinoxylan such as grain-based materials, in the gluten-starch separation industry of wheat, in the production of syrups derived from starch, in the production of biofuels and the like.

STATEMENTS OF THE INVENTION

[0054] In a first aspect, the present invention provides an isolated polypeptide having xylanase activity, selected from the group consisting of:

[0055] (a) a polypeptide comprising an amino acid sequence having at least 87% identity (suitably at least 89%, suitably at least 90%, suitably at least 92%, suitably at least 94%, suitably suitably at least 98%, suitably at least 100% identity) with SEQ ID NO: 1;

[0056] (b) a polypeptide encoded by a polynucleotide that has at least 87% identity (suitably, at least 89%, suitably, at least 90%, suitably, at least 92%, suitably, at least 94%, suitably , at least 98%, suitably at least 100% identity) with SEQ ID NO:2; or

[0057] (c) a fragment of a polypeptide of a) or b) which fragment has xylanase activity.

[0058] In a further aspect, the present invention provides an isolated polynucleotide (e.g., cDNA) comprising a nucleotide sequence encoding the polypeptide of the present invention.

[0059] In yet another aspect, there is provided an isolated polynucleotide (e.g., cDNA) comprising a polynucleotide that has at least 87% identity (suitably at least 89%, suitably at least 90%, suitably at least 92 %, suitably at least 94%, suitably at least 98%, suitably at least 100% identity) with SEQ ID No: 2; or an isolated polynucleotide that differs from SEQ ID NO: 2 due to degeneracy of the genetic code.

[0060] The present invention further provides an isolated polynucleotide (e.g. cDNA) selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, or an isolated polynucleotide that differs from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO : 12 due to the degeneration of the genetic code.

[0061] In one aspect, the present invention provides a nucleic acid construct comprising the polynucleotide of the present invention operably linked to one or more control sequences that direct production of the polypeptide in an expression host.

[0062] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid construct of the present invention.

[0063] Additionally, there is provided a recombinant host cell comprising a polynucleotide according to the present invention, a nucleic acid construct of the present invention or a vector according to the present invention.

[0064] The present invention further provides a method for producing the polypeptide of the present invention, comprising (a) culturing a host cell comprising a nucleic acid construct according to the present invention under conditions suitable for producing the polypeptide; and (b) recovering the polypeptide.

[0065] In another aspect of the present invention, a fermentate produced by the method of the present invention is provided.

[0066] Yet another aspect of the present invention is the provision of a xylanase produced by the method of the present invention.

[0067] The present invention additionally provides an enzyme composition comprising a) a synthetic xylanase according to the present invention, b) the fermentate according to the present invention, or c) a combination thereof.

[0068] The present invention further provides a feed additive composition comprising a) a synthetic xylanase enzyme according to the present invention, b) the fermentate according to the present invention, or c) a combination thereof.

[0069] In another aspect of the present invention there is provided a premix comprising a) a synthetic xylanase enzyme according to the present invention, b) the fermentate according to the present invention, c) the enzyme composition according to with the present invention, d) a feed additive composition according to the present invention or e) a combination thereof; and at least one vitamin and/or at least one mineral.

[0070] The present invention further provides a feed (or animal feed) comprising a) a synthetic xylanase enzyme according to the present invention, b) the fermentate according to the present invention, c) the enzyme composition according to with the present invention, d) a feed additive composition according to the present invention, e) a premix according to the present invention, or f) a combination thereof.

[0071] In a further aspect there is provided a method of preparing a feed comprising admixing a feed component with a) a synthetic xylanase according to the present invention, b) the fermentate according to the present invention, c) the enzyme composition according to the present invention, d) a feed additive composition according to the present invention, e) a premix according to the present invention or f) a combination thereof.

[0072] The present invention further provides a method for degrading arabinoxylan-containing material into a xylan-containing material, comprising admixing said xylan-containing material with a) a synthetic xylanase enzyme according to the present invention, b) the fermentate according to with the present invention, c) the enzyme composition according to the present invention, d) a feed additive composition according to the present invention, e) a premix according to the present invention or f) a combination of the same.

[0073] In another aspect, there is provided the use of a) a synthetic xylanase enzyme according to the present invention, b) the fermentate according to the present invention, c) the enzyme composition according to the present invention, d) a feed additive composition according to the present invention, e) a premix according to the present invention, or f) a combination thereof for solubilizing arabinoxylan in a xylan-containing material.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0074] Except where otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this description pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ED., John Wiley and Sons, New York, USA (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY, USA (1991) ) provide a person skilled in the art with a general dictionary of many of the terms used in this description.

[0075] This disclosure is not limited to the exemplary methods and materials described herein, and any methods and materials similar or equivalent to those described herein may be used in practicing or testing the embodiments of the present disclosure. Numeric ranges include the numbers that define the range. Unless otherwise noted, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written from left to right in amino to carboxy orientation, respectively.

[0076] The titles given herein are not limitations of the various aspects or modalities of the description that can be obtained by reference to the descriptive report as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0077] Amino acids are designated herein using either the amino acid name, the three-letter abbreviation, or the one-letter abbreviation.

[0078] The term "protein", as used herein, includes proteins, polypeptides and peptides.

[0079] As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme".

[0080] The terms "protein" and "polypeptide" are used interchangeably in the present invention. In the present specification and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) IUPAC-IUB. It should also be understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0081] Other definitions of terms may appear throughout the descriptive report. Before the exemplary embodiments are described in detail, it should be understood that this description is not limited to the specific embodiments described, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present description will be limited only by the appended claims.

[0082] Where a range of values is provided, it is understood that each intermediate value, up to the tenth of a unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range, is also specifically described . Each lesser range between any specified value or intermediate value within a specified range and any other specified or intermediate value within that specified range falls within this description. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range where either, neither, or both limits are included in the smaller ranges is also covered within this description, subject to any limits specifically excluded in the specified range. . Where the specified range includes one or both of these boundaries, ranges excluding one or both of these included boundaries are also included in the present description.

[0083] It should be noted that, as used herein and in the appended claims, the singular forms of "an", "an", and "the/the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such candidate agents, and reference to "a feed" includes reference to one or more feeds and their equivalents known to those skilled in the art, and so forth.

[0084] The publications discussed herein are provided for your description only prior to the filing date of this application. Nothing therein should be construed as an admission that such publications constitute prior art to the appended claims.

[0085] The increase in prices of the raw material traditionally used as a source of energy in animal feed, as a raw material in the production of biofuels, as an ingredient in the production of beer or malting, or as a raw material in processes of The separation of starch and gluten from wheat, for example, has resulted in the inclusion of low-cost fibrous materials in starting substrates for these industries, particularly the use of low-cost fibrous by-products in animal feed.

[0086] The addition of fiber can cause several disadvantageous effects. For example, in animal feed, the addition of fiber can cause anti-nutritional effects. In animal feeds, hemicellulose and cellulose (including insoluble arabinoxylan) also form physical barriers that encapsulate (or trap) nutrients such as starch and protein and thus prevent the animal from gaining access to these nutrients.

[0087] Hemicellulose and cellulose (including insoluble arabinoxylans (AXinsol)) are also potential sources of energy, as they consist of C5 and C6 saccharides. C6 monosaccharides can be used as an energy source by the animal, while C5 oligosaccharides can be transformed into short-chain fatty acids by the microflora present in the animal's gastrointestinal tract (van den Broek et al., 2008 Molecular Nutrition & Food Research, 52, 146-63), and these short-chain fatty acids can be absorbed and digested by the animal's gastrointestinal tract.

[0088] The release of nutrients from animal feeds as a result of physical barrier degradation depends on the ability of xylanase to degrade insoluble fiber components (eg, insoluble arabinoxylans (AXinsol)).

[0089] In a first aspect, the present invention provides an isolated polypeptide having xylanase activity, selected from the group consisting of:

[0090] (a) a polypeptide comprising an amino acid sequence that has at least 87% identity with SEQ ID NO: 1;

[0091] (b) a polypeptide encoded by a polynucleotide that has at least 87% identity with SEQ ID NO: 2 or

[0092] (c) a fragment of a polypeptide of a) or b) which fragment has xylanase activity.

[0093] In another aspect, the present invention provides an isolated polypeptide having xylanase activity, selected from the group consisting of:

[0094] (a) a polypeptide comprising an amino acid sequence that has at least 89% identity with SEQ ID NO: 1;

[0095] (b) a polypeptide encoded by a polynucleotide that has at least 89% identity with SEQ ID NO: 2 or

[0096] (c) a fragment of a polypeptide of a) or b) which fragment has xylanase activity.

[0097] In one embodiment, the present invention provides an isolated polypeptide having xylanase activity, selected from the group consisting of:

[0098] (a) a polypeptide comprising an amino acid sequence that has at least 90% identity with SEQ ID NO: 1;

[0099] (b) a polypeptide encoded by a polynucleotide that has at least 90% identity with SEQ ID NO: 2 or

[00100] (c) a fragment of a polypeptide of a) or b) which fragment has xylanase activity.

[00101] In another embodiment, the present invention provides an isolated polypeptide having xylanase activity, selected from the group consisting of:

[00102] (a) a polypeptide comprising an amino acid sequence that has at least 94% identity with SEQ ID NO: 1;

[00103] (b) a polypeptide encoded by a polynucleotide that has at least 94% identity with SEQ ID NO: 2 or

[00104] (c) a fragment of a polypeptide of a) or b) which fragment has xylanase activity.

[00105] In yet another embodiment, the present invention provides an isolated polypeptide having xylanase activity, selected from the group consisting of:

[00106] (a) a polypeptide comprising an amino acid sequence that has at least 98% identity with SEQ ID NO: 1;

[00107] (b) a polypeptide encoded by a polynucleotide that has at least 98% identity with SEQ ID NO: 2 or

[00108] (c) a fragment of a polypeptide of a) or b) which fragment has xylanase activity.

[00109] The polypeptide according to the present invention may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO : 9 and SEQ ID NO: 11.

[00110] In one embodiment, the present invention provides a polypeptide that has xylanase activity, comprising an amino acid sequence that has at least 87% identity with one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11.

[00111] In one embodiment, the present invention provides a polypeptide that has xylanase activity, comprising an amino acid sequence that has at least 93% identity with one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO:11.

[00112] In one embodiment, the present invention provides a polypeptide that has xylanase activity, comprising an amino acid sequence that has at least 98% identity with one of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11.

[00113] In one embodiment, the polypeptide having xylanase activity comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11.

[00114] In a specific embodiment, the polypeptide having xylanase activity comprises an amino acid sequence shown in the present invention as SEQ ID NO: 1.

[00115] In yet another embodiment, the polypeptide according to the present invention may consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO : 7, SEQ ID NO: 9 and SEQ ID NO: 11.

[00116] In one embodiment, the polypeptide can be encoded by a polynucleotide that has at least 87% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12; or at least 87% identity with a polynucleotide that differs from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 due to degeneration of the genetic code.

[00117] In another embodiment, the polypeptide can be encoded by a polynucleotide that has at least 93% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12; or at least 93% identity with a polynucleotide that differs from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 due to degeneration of the genetic code.

[00118] In another embodiment, the polypeptide can be encoded by a polynucleotide that has at least 95% identity with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12; or at least 95% identity with a polynucleotide that differs from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 due to degeneration of the genetic code.

[00119] In a preferred embodiment, the polypeptide of the present invention is encoded by a polynucleotide selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12; or a polynucleotide that differs from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 due to degeneracy of the genetic code.

[00120] In one embodiment, the polypeptide of the present invention is encoded by a polynucleotide comprising the nucleotide sequence shown in the present invention as SEQ ID NO: 2, or a polynucleotide that differs from SEQ ID NO: 2 due to the degeneracy of the genetic code .

[00121] Suitably, the nucleic acid or polynucleotide sequences taught in the present invention may be genomic DNA, synthetic DNA or RNA.

[00122] In one embodiment, the nucleic acid or polynucleotide sequences taught in the present invention can be DNA, more preferably cDNA.

[00123] In one embodiment, the present invention provides a nucleic acid construct comprising the polynucleotide of the present invention operably linked to one or more control sequences that direct production of the polypeptide in an expression host.

[00124] Also contemplated is a recombinant expression vector comprising the nucleic acid construct of the present invention or polynucleotides of the present invention; and host cells (e.g., recombinant host cells) comprising the nucleic acid construct of the present invention or polynucleotides of the present invention; or vectors according to the present invention.

[00125] In one embodiment there is taught a method for producing the polypeptide of the present invention, comprising (a) culturing a host cell comprising a nucleic acid construct of the present invention under conditions suitable for producing the polypeptide; and (b) recovering the polypeptide.

[00126] The polypeptide produced in this way can be recovered. The polypeptide so produced can be used as part of a fermentate or can be isolated and/or purified to produce an isolated or purified synthetic xylanase.

[00127] In a preferred embodiment, the synthetic xylanase produced according to a method of the present invention is recovered.

[00128] In a preferred embodiment, the synthetic xylanase produced according to a method of the present invention is isolated and/or purified.

[00129] In some embodiments, the synthetic xylanase can be used directly as a fermentate, without isolation and/or purification of the enzyme.

[00130] The host cell of the present invention can be selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a filamentous fungal cell and a plant cell. Preferably, the host cell is a bacterial or fungal cell.

[00131] In one embodiment, preferably the synthetic xylanase is an endoxylanase, for example an endo-1,4-β-d-xylanase. The classification for an endo-1,4-β-d-xylanase is E.C. 3.2.1.8.

[00132] In a preferred embodiment, the polypeptide of the present invention has at least 40% residual activity of the xylanase activity after incubation at 65°C for 10 minutes at pH 6.

[00133] In a preferred embodiment, the polypeptide of the present invention has at least 50% residual activity of the xylanase activity after incubation at 65°C for 10 minutes at pH 6.

[00134] In another preferred embodiment, the polypeptide of the present invention has at least 80% residual activity of the xylanase activity after incubation at 61°C for 10 minutes at pH 6.

[00135] Suitably, the polypeptide of the present invention has a residual activity of at least 70% when incubated for two hours with 0.2 mg/ml pepsin in a buffer solution of pH 3.5 at a temperature of 40°C.

[00136] In a preferred embodiment, the polypeptide of the present invention has at least 60% residual xylanase activity after a feed comprising the polypeptide has been treated with dry steam to reach a target temperature of 90°C after 30 seconds.

[00137] In a preferred embodiment, the polypeptide of the present invention has at least 60% residual xylanase activity after a feed comprising the polypeptide has been conditioned for 30 seconds at 90°C, for example, as part of a pelleting process .

[00138] In a preferred embodiment, the synthetic xylanase according to the present invention has a Tm value greater than 61°C (preferably greater than 65°C, preferably greater than 69°C, preferably greater than 73° C), where the Tm value is measured as the temperature at which 50% of residual activity is obtained after 10 min of incubation.

[00139] The thermostability of the synthetic xylanase according to the present invention can be determined using the "Assay for the measurement of thermostability" (see below).

Test for measuring thermostability

[00140] The thermal denaturation profiles of synthetic xylanases were measured by diluting and pre-incubating the enzyme samples in 25 mM MES buffer, pH 6.0, for 10 min at varying temperatures (for example, 61, 65, 69 and 73°C, respectively) and subsequent measurement of residual activity by the xylanase activity assay described in Example 2. The activity measured without pre-incubation was defined as 100% and the residual activity of each synthetic xylanase at each temperature was calculated in relation to this. The Tm value is calculated from thermal denaturation profiles as the temperature at which 50% of residual activity is obtained.

[00141] The complete details of the test for measuring thermostability can be found in Example 2 (see "Thermostability test").

[00142] The residual activity of each synthetic xylanase was calculated as the ratio between the activity measured in the enzyme samples subjected to stress (heat-treated) and not subjected to stress (not heat-treated), respectively: (Average relative activity of sample under stress)/(Average relative activity of sample without stress).

[00143] In one embodiment, a synthetic xylanase is considered thermostable, according to the present invention, if it has a Tm value above 65°C, where the Tm value is the temperature at which 50% of residual activity is obtained after 10 minutes of incubation. The Tm value can be measured according to the thermostability measurement test as taught here.

[00144] In one embodiment, a synthetic xylanase is considered thermostable, according to the present invention, if it has a Tm value above 69°C, where the Tm value is the temperature at which 50% of residual activity is obtained after 10 minutes of incubation. This Tm value can be measured according to the thermostability measurement test, as taught above.

[00145] In one embodiment, a synthetic xylanase is considered thermostable, according to the present invention, if it has a Tm value above 73°C, where the Tm value is the temperature at which 50% of residual activity is obtained after 10 minutes of incubation. The Tm value can be measured according to the thermostability measurement test as taught here.

[00146] A surprising technical advantage of the synthetic xylanase (or the composition comprising the synthetic xylanase) is its remarkable ability to withstand heat treatment (e.g. during the pelletizing process) of up to about 85°C (suitably, of up to about 90°C). Heat treatment can be carried out for 30 seconds. Withstanding such a heat treatment means that at least about 40%, suitably at least 50%, of the enzyme that was present/active in the additive before heating to the specified temperature is still present/active after cooling to room temperature. Preferably, at least about 60% (suitably at least about 70%, suitably at least about 80%) of the enzyme that is present and active in the additive before heating to the specified temperature is still present and active after the additive. cool down to room temperature.

[00147] The term "thermostability" is the ability of an enzyme to resist irreversible inactivation (generally by denaturation) at a relatively high temperature. This means that the enzyme retains a specified amount of enzyme activity after exposure to an identified temperature for a specified period of time.

[00148] There are many ways to measure thermostability. By way of example, enzyme samples can be incubated without the substrate for a defined period of time (e.g. 10 min or 1 to 30 min) at an elevated temperature compared to the temperature at which the enzyme is stable for a long time (days). After incubation at elevated temperature, the enzyme sample is analyzed for residual activity at the permissive temperature, eg 30°C (alternatively 25 to 50°C or even 70°C). Residual activity is calculated as relative to an enzyme sample that has not been incubated at an elevated temperature.

[00149] The thermostability can also be measured as enzyme inactivation as a function of temperature. Here, enzyme samples are incubated without substrate for a defined period of time (e.g. 10 minutes or 1 to 30 minutes) at various temperatures and after incubation are tested for residual activity at the permissive temperature of e.g. , 30°C (alternatively 25 to 70°C or even higher). Residual activity at each temperature is calculated relative to an enzyme sample that was not incubated at an elevated temperature. The resulting thermal denaturation profile (residual activity as a function of temperature) can be used to calculate the temperature at which 50% of the residual activity is obtained. This value is defined as the Tm value.

[00150] Still additionally, thermostability can be measured as enzyme inactivation as a function of temperature. Here, enzyme samples are incubated without substrate at a defined elevated temperature (e.g. 76°C) for various periods of time (e.g. between 10 seconds and 30 minutes) and after incubation are tested for residual activity. at the permissive temperature of, for example, 30°C (alternatively 25 to 70°C or even higher). Residual activity at each temperature is calculated relative to an enzyme sample that was not incubated at an elevated temperature. The resulting inactivation profile (time versus residual activity) can be used to calculate the temperature at which 50% of residual activity is obtained. This is normally given as T1/2.

[00151] These are examples of how to measure thermostability. Thermostability can also be measured by other methods. Preferably, thermostability is determined using the "Assay for measuring thermostability" as taught herein.

[00152] As opposed to thermostability, thermoactivity is the activity of the enzyme as a function of temperature. To determine thermoactivity, enzyme samples can be incubated (tested) for the time period defined by the assay at various temperatures in the presence of substrate. Enzyme activity is obtained during or immediately after incubation, depending on the assay (eg, reading an OD value that reflects the amount of reaction product formed). The temperature at which the highest activity is obtained is the temperature optimum of the enzyme for the given assay conditions. The activity obtained at each temperature can be calculated in relation to the activity obtained at the optimum temperature. This will provide a temperature profile for the enzyme under the given assay conditions.

[00153] In the present application, thermostability is not the same as thermoactivity.

[00154] In some embodiments, the enzyme that has xylanase activity, for example, the GH10 xylanase enzyme (such as modified GH10 xylanase enzyme) or a fragment thereof, according to the present invention, must have good xylanase activity at a pH between about 5 and 6.

[00155] Preferably, the enzyme having xylanase activity, for example, GH10 xylanase enzyme (such as modified GH10 xylanase enzyme) or a fragment thereof, according to the present invention, retains more than 70% of the activity maxima between pH4 and 8, suitably between pH 4.6 and 7.

[00156] In some embodiments, for example, in feed applications, the enzyme having xylanase activity, for example, GH10 xylanase enzyme (such as modified GH10 xylanase enzyme) or a fragment thereof, according to the present invention , preferably retains more than 70% of the maximum activity at a pH between 4.9 and 6.

[00157] Without wishing to be bound by theory, pH can also have an important effect on enzyme effectiveness and efficiency. For feed applications, in particular the pH profile of the xylanases of the present invention favors activity in the small intestine under neutral conditions.

[00158] The present invention further provides an enzyme composition or a feed additive composition comprising the polypeptide of the present invention or the fermented according to the invention.

[00159] The present invention further provides a premix comprising a polypeptide of the present invention or the fermented one according to the present invention, or the enzyme composition of the present invention, or a feed additive composition according to the present invention and/or at least one mineral.

[00160] In some embodiments, the feed additive composition according to the present invention or the premix according to the present invention further comprises one or more enzymes selected from the group consisting of a protease ( for example, subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)) and/or an amylase (including α -amylases (E.C. 3.2.1.1), G4-forming amylases (E.C. 3.2.1.60), β-amylases (E.C. 3.2.1.2) and Y-amylases (E.C. 3.2.1.3)).

[00161] The synthetic xylanase according to the present invention or a fermentate comprising the same, or an enzyme composition comprising the same can be used in a method to degrade arabinoxylan-containing material into a xylan-containing material.

[00162] Suitably, the arabinoxylan may be insoluble arabinoxylan (AXinsol).

[00163] In one embodiment, the xylan-containing material is selected from one or more of the group consisting of: an animal feed or feed; a feed component; a grain-based material; a porridge; the wort; a malt; malted barley; an auxiliary compound, a barley porridge and a cereal flour.

[00164] In one embodiment, the arabinoxylans are solubilized without increasing the viscosity of the reaction medium.

[00165] In one embodiment of the present invention, the feed or animal feed or feed component comprises or consists of corn, DDGS (such as cDDGS), wheat, wheat bran or a combination thereof.

[00166] In a preferred embodiment, the feed or food for animals is a corn-based animal feed.

[00167] The synthetic xylanase according to the present invention can be used in combination with one or more of the enzymes selected from the group consisting of: endoglucanases (E.C. 3.2.1.4); celiobiohydrolases (E.C. 3.2.1.91), β-glucosidases (E.C. 3.2.1.21), cellulases (E.C. 3.2.1.74), lichenases (E.C. 3.2.1.73), lipases (E.C. 3.1.1.3), lipid acyltransferases (generally classified as E.C. 2.3 .1.x), phospholipases (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), phytases (e.g. 6-phytase (E.C. 3.1.3.26) or a 3-phytase (E.C. 3.1.3.8), amylases, alpha-amylases (E.C. 3.2.1.1), other xylanases (E.C. 3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.2.1.72, E.C. 3.2.1.136), glycoamylases (E.C. 3.2.1.3), hemicells es , proteases (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)), debranching enzyme, cutinases , esterases and/or mannanases (e.g. a β-mannanase (E.C. 3.2.1.78)).

[00168] The synthetic xylanase according to the present invention can be used in combination with one or more of the enzymes selected from the group consisting of a protease (for example, subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4 .24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)) and/or an amylase (including α-amylases (E.C. 3.2.1.1), G4-forming amylases (E.C. 3.2 .1.60), β-amylases (E.C. 3.2.1.2) and Y-amylases (E.C. 3.2.1.3)).

[00169] In one embodiment, the method or use according to the present invention comprises administering to a subject a synthetic xylanase enzyme according to the present invention, or a fermentate comprising a synthetic xylanase enzyme according to the present invention, or an enzyme composition comprising a synthetic xylanase enzyme according to the present invention, or a feed additive composition comprising a synthetic xylanase enzyme according to the present invention, or a premix comprising a synthetic xylanase enzyme according to the present invention, or an animal feed comprising a synthetic xylanase enzyme according to the present invention.

[00170] In one embodiment, the method or use of the present invention is (or forms part of) a process for separating starch and gluten from wheat.

[00171] In another embodiment, the method or use of the present invention is (or forms part of) a process for producing biofuels (eg, bioethanol) or biochemical agents (eg, biobased isoprene).

[00172] In another embodiment, the method or use of the present invention is (or forms part of) a malting or brewing process.

[00173] Suitably, a fermented beverage, for example beer, produced by a method according to the present invention is intended by the present invention.

[00174] Both the polypeptide sequences and the nucleic acid sequences taught herein are preferably isolated.

[00175] The synthetic xylanase of the present invention is preferably a GH10 xylanase. In other words, the xylanase can have a molecular weight in the range of 32 to 39 kDa and/or the catalytic domain of the xylanase consists of an eightfold β/α cylindrical structure (as shown in Harris 19961996 - Acta. Crystallog. Sec. D 52 , 393-401).

[00176] In one aspect of the invention, the xylanase of the invention is a xylanase of the glycoside hydrolase (GH) family 10. The term "glucoside hydrolase (GH) family 10" means that the xylanase in question is or may be classified in the GH family 10.

[00177] Protein similarity searches (for example, the BLAST tool at http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=Blast Home) can determine whether an unknown sequence fits the term "member of the GH10 xylanase family"; particularly, GH families can be classified based on sequence homology in major regions. Additionally or alternatively, to determine whether an unknown protein sequence is a GH10 family xylanase protein, the assessment can be made, not only on the basis of similarity/homology/identities, but also on the similarity of the 3D structure. Classification of GH families is often based on 3D bending. The software that predicts the 3D folding of an unknown protein sequence is HHpred (http://toolkit.tuebingen.mpg.de/hhpred). The power of this protein structure prediction software is in identifying homologous sequences with known structure to be used as a model. This works well because structures diverge much more slowly than primary sequences. Proteins in the same family can have very similar structures, even when their sequences have diverged beyond recognition.

[00178] In practice, an unknown string can be pasted into the software (http://toolkit.tuebingen.mpg.de/hhpred) in FASTA format. Having done this, the quest can be sent. The search result will show a list of sequences with known 3D structures. To confirm that the unknown sequence is indeed a GH10 xylanase, GH10 xylanases can be found within the homologous list having a probability of >90. Not all proteins identified as homologous will be characterized as GH10 xylanases, but some will. The latter are proteins with a known structure and biochemical characterization that identifies them as xylanases. The former were not characterized as biochemically as GH10 xylanases. Several references describe this protocol, such as Soding J. (2005) Protein homology detection by HMM-HMM comparison - Bioinformatics 21, 951-960 (doi:10.1093/bioinformatics/bti125) and Soding J, Biegert A, and Lupas AN. (2005) The HHpred interactive server for protein homology detection and structure prediction - Nucleic Acids Research 33, W244--W248 (web server edition) (doi:10.1093/nar/gki40).

[00179] According to the Cazy website (http://www.cazy.org/), family 10 glycoside hydrolases can be characterized as follows:

[00180] Known activities: endo-1,4-β-xylanase (EC 3.2.1.8); endo-1,3-β-xylanase (EC 3.2.1.32); tomatinase (EC 3.2.1.-)

[00181] Mechanism: Retention

[00182] Clan: GH-A

[00183] Catalytic/base nucleophile: Glu (experimental)

[00184] Catalytic proton donor: Glu (experimental)

[00185] Status of 3D structure: (β / α)8

[00186] The GH10 xylanase of the present invention may have a catalytic domain with molecular weights in the range of 32 to 39 kDa. The structure of the GH10 xylanase catalytic domain of the present invention consists of an eight-fold β/α barrel (Harris et al. 1996 - Acta. Crystallog. Sec. D 52, 393-401).

[00187] Three-dimensional structures are available for a large number of enzymes of the GH10 family, and the first to be solved were those of xylanase A from Streptomyces lividans (Derewenda et al. J Biol Chem 1994 Aug 19; 269(33) 20811-4) , Cex endoglucanase from C. fimi (White et al. Biochemistry 1994, Oct 25; 33(42) 12546-52) and Xyn10A from Cellvibrio japonicus (formerly known as Pseudomonas fluorescens subsp. xylanase A) (Harris et al. Structure 1994 Nov 15; 2(11) 1107-16.). As members of the GHA clan, they have a classic barrel fold (α/β)8 TIM with the two key active site glutamic acids located at the C-terminal ends of the beta 4 (acid/base) and 7 (nucleophile) strands ( Henrissat et al Proc Natl Acad Sci U S A 1995 Jul 18;92(15) 7090-4).

[00188] The term "GH10 xylanase" as used herein means a polypeptide having xylanase activity and having a TIM barrel fold (α/β) 8 with the two main active site glutamic acids situated at the C-terminus of the ends of beta chains 4 (acid/base) and 7 (nucleophile).

[00189] In one embodiment, the synthetic xylanase according to the present invention is capable of degrading (or degrades) a xylan-containing material, particularly arabinoxylans, and particularly insoluble arabinoxylans (AXinsol).

[00190] The term "consisting essentially of", as used herein, means that unspecified components may be present if the characteristics of the claimed composition are not materially affected.

[00191] The term "consisting of" means that the proportions of specific ingredients must total 100%.

[00192] The term "comprising" used in the present invention can be changed in some embodiments to refer to "consists essentially of" or "consists of" (both having a more limited meaning than "comprising").

[00193] In one embodiment, the insoluble arabinoxylan-containing material is not wheat straw.

[00194] The term "fragment thereof", as used herein, means an active fragment. In other words, the fragment is one that has xylanase activity. Suitably, the fragment may have the same xylanase activity as the full synthetic xylanase enzyme from which the fragment is derived. Alternatively, the fragment may have modified activity (eg, enhanced specificity, specific activity, pH or temperature profile) compared to the synthetic xylanase from which the fragment is derived. Furthermore, the fragment must retain the thermostable properties of the synthetic xylanase of which it is a fragment.

[00195] In one embodiment, the fragment is at least 60% of the total length of the synthetic xylanase from which the fragment is derived.

[00196] In one embodiment, the fragment is at least 75% of the total length of the synthetic xylanase from which the fragment is derived.

[00197] In one embodiment, the fragment is at least 85% of the total length of the synthetic xylanase from which the fragment is derived.

[00198] In one embodiment, the fragment is at least 95% of the total length of the synthetic xylanase from which the fragment is derived.

[00199] In one embodiment, the fragment is at least 98% of the total length of the synthetic xylanase from which the fragment is derived.

[00200] In one embodiment, the fragment is a fragment of one or more of the sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11.

[00201] In one embodiment, the synthetic xylanase according to the present invention a) comprises one of the amino acid sequences presented here as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9 or SEQ ID NO: 11, or b) comprises an amino acid sequence that has at least 96%, preferably at least 98.5%, preferably at least 99% identity with the amino acid sequences presented herein as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11, or c) comprises a fragment corresponding to at least 85% full length synthetic xylanase shown here as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

Uses

[00202] The synthetic xylanase of the present invention can be suitably used in any of the following applications:

[00203] a) An additive in animal feed and/or

[00204] b) A feed supplement for an animal and/or

[00205] c) Degradation of grain-based material (for example, this can be whole grain or part grain). Degradation products (e.g. glucose) can be used as a feedstock for any fermentation process, such as in the production of biofuels (e.g. bioethanol) or in the production of other products such as biochemicals (e.g. biobased isoprene) . Therefore, in one embodiment, the present invention relates to the production of biofuels (eg, bioethanol) and the increased use of grain-based material in the biofuel industry; and/or

[00206] d) Gluten and starch separation industry from cereals (eg wheat). The resulting product(s) may be starch (e.g. purified starch) and/or gluten and/or fiber and/or water solubles (such as soluble pentosans). In one embodiment, the present invention relates to the production of starch and/or gluten and/or

[00207] e) Improvement of malting and brewing, for example, by decomposition of grain-based material (for example, malted barley) and/or

[00208] f) decomposition of AXsol or AXinsol decomposition products, to ensure that the viscosity is not increased and/or the viscosity is reduced in the reaction mixture; and/or

[00209] g) reduce viscosity when degrading grain-based materials, for example, in biofuel production processes (eg bioethanol).

[00210] In one embodiment, the synthetic xylanase of the present invention is used in an animal feed. Preferably, an animal feed comprising maize is a maize-based animal feed.

[00211] In one embodiment, the synthetic xylanase of the present invention is used in malting or brewing.

[00212] In another embodiment, the synthetic xylanase of the present invention is used in wheat starch-gluten separation.

[00213] In yet another embodiment, the synthetic xylanase of the present invention is used in the decomposition of grain-based material and can be part of the process of producing biofuels (eg, bioethanol).

Benefits

[00214] The innovative synthetic xylanase taught in the present invention has many advantages compared to known xylanases.

[00215] Furthermore, the synthetic xylanase of the present invention is particularly thermostable. This provides significant advantages in some applications. In particular, in feed applications, the enzymes may be subjected to heat treatment, for example during pelleting processes. Thus, enzymes need to be able to maintain their activity after such processing. The synthetic xylanases of the present invention are particularly unexpectedly thermostable. Specifically, the synthetic xylanase of the present invention has been found to have very high recovery (e.g. residual activity) after the pelletizing process.

[00216] Suitably, the synthetic xylanase has a Tm value of more than 65°C, where the Tm value is the temperature at which 50% of residual activity is obtained after 10 minutes of incubation.

[00217] In addition, improved thermostability is also very beneficial during starch degradation, which occurs at elevated temperatures during liquefaction (about 85 to 95°C). The fact that it is thermostable allows the addition of the enzyme during this step.

[00218] Additionally, or alternatively, synthetic xylanases have been found to have unexpectedly high resistance to pepsin.

[00219] Pepsin is a digestive protease excreted by the animal in the first part of the digestive system. Pepsin degrades the protein, making it available as a nutrient for the animal. Exogenous enzymes, ie enzymes added to the feed, are also proteins and they will be degraded if they are susceptible to degradation by pepsin. This will, in most cases, destroy enzyme activity. Therefore, it is highly advantageous for synthetic xylanases to have pepsin resistance.

[00220] The synthetic xylanases of the present invention, as taught here, are also unexpectedly good at solubilizing pentosans.

[00221] The synthetic xylanases of the present invention, as taught here, are also unexpectedly good at solubilizing AXinsol.

[00222] Surprisingly, it has been found that the synthetic xylanase of the present invention is particularly good at degrading xylan-containing materials such as arabinoxylans, for example, AXinsol, on a wide spectrum of substrates, corn, wheat, DDGS, etc., in particular, corn and corn-based substrates, in particular wheat (including wheat-based) or corn (including corn-based) products. This is in contrast to previously known enzymes, which are often inferior in solubilizing AXinsol on corn or corn based substrates or which are not as efficient on any of the wheat and corn based substrates.

[00223] Furthermore, the synthetic xylanase of the present invention may be particularly suitable not only for decomposing (solubilizing) AXinsol, but also for effectively decomposing (or degrading) the solubilized polymers. By being able to efficiently (quickly) decompose (degrade) the solubilized polymers (obtained from the dissolution of AXinsol) a reduction in viscosity is obtained. This last effect is essential in some of the claimed applications.

[00224] Typically, conventional xylanases can decompose AXinsol, but this will lead to an increase in the production of polymeric products which will lead to an increase in the viscosity of the mixture. This higher viscosity is disadvantageous in many applications.

[00225] It was found that the synthetic xylanases of the present invention, as described herein, not only decompose (solubilize) insoluble arabinoxylans (AXinsol) from a wide range of substrates, including corn, wheat, DDGS, etc., in particular corn and corn-based substrates, in particular both wheat (including wheat-based) and corn (including corn-based) products, but also efficiently decompose the polymers thus solubilized to ensure that the viscosity does not increase and/or to reduce viscosity.

[00226] In some embodiments, the synthetic xylanases of the present invention, as described herein, are capable of degrading AXsol or the decomposition products of AXinsol, to ensure that the viscosity does not increase and/or that the viscosity is reduced in the reaction mixture .

[00227] In particular, the synthetic xylanases of the present invention are specifically effective in degrading xylan-containing materials, such as arabinoxylans, e.g., AXinsol, in maize and maize-based substrates.

[00228] Many of the xylanases marketed for use in animal feeds to solubilize pentosans are GH11 enzymes. It was considered by those skilled in the art that GH10 xylanases were not as strong in solubilizing pentosans, particularly AXinsol, compared to GH11 xylanases. Surprisingly, the synthetic xylanases disclosed here, which are GH10 xylanases, have been found to be particularly suitable for solubilizing AXinsol on a broad spectrum of substrates, including corn-based substrates. Surprisingly, the present inventors have found that the synthetic xylanases of the present invention (and taught herein) outperform commercial GH11 xylanases in terms of their ability to solubilize pentosans.

[00229] The fact that synthetic xylanases effectively solubilize AXinsol extracted from corn and corn-based substrates is significantly advantageous, as corn contains much more AX in insoluble form than other cereals such as wheat and rye , for example. Therefore, only xylanases that can decompose AXinsol can show significant benefit for animals fed a corn and soy based diet, for example.

[00230] It was completely unexpected that a GH10 xylanase would solubilize AXinsol so well in cereals, particularly corn or corn-based substrates.

[00231] The synthetic xylanases of the present invention are capable of effectively (and rapidly) degrading polymers and/or oligomers that are produced from the solubilization of AXinsol or that are present in grain-based materials. This leads to an unexpected advantage for the synthetic xylanases taught in the present invention, in that they are particularly good in various applications for keeping viscosity low or for reducing viscosity, for example in animal feed; in the manufacture of fermented beverage and/or in malting; in the production of grain-based glucose, eg to further process biofuels and/or biochemicals (e.g. bioisoprene); or in the gluten-wheat starch separation industry for the production of, for example, starch.

[00232] An advantage of the present invention is that it improves the separation of starch and wheat gluten.

[00233] The enzyme of the present invention is particularly effective in improving the performance of an individual or improving the digestibility of a feedstock in a feed and/or for improving feed efficiency in an individual.

Material containing xylan

[00234] The synthetic xylanase of the present invention (or a composition comprising the synthetic xylanase of the present invention) can be used to degrade any xylan-containing material.

[00235] In one embodiment, the xylan-containing material is any plant material comprising arabinoxylan.

[00236] In one embodiment, the xylan-containing material is any plant-derived material comprising insoluble arabinoxylan (AXinsol).

[00237] In one embodiment, the xylan-containing material is a food or feed component.

[00238] In one embodiment, the xylan-containing material is a grain-based material (including whole grain or partial grain or malted grain, e.g., malted barley). When the method is related to the production of biofuels (eg bioethanol production) then preferably the xylan-containing material is a grain-based material.

[00239] In another embodiment, the xylan-containing material may be a barley malt or gruel, or malted barley, or combinations thereof.

[00240] In yet another further embodiment, the xylan-containing material can be a cereal flour (for example, wheat, oat, rye or barley flour). When the method is concerned with a starch and gluten separation process, preferably the xylan-containing material is a cereal flour (eg wheat, oat, rye or barley flour).

Decomposition or degradation

[00241] The enzyme (or composition comprising the enzyme) of the present invention, or as disclosed in the present invention, can be used to decompose (degrade) AXinsol or AXinsol degradation products.

[00242] The term "decompose" or "degrade" is synonymous with hydrolyze.

Solubilization/degradation

[00243] The present invention relates to a method for degrading a xylan-containing material (preferably an arabinoxylan-containing material, preferably an insoluble arabinoxylan-containing material (AXinsol)) to produce soluble pentosans (which can be polymeric, oligomeric or monomeric).

[00244] This method can be described here as pentosan solubilization or arabinoxylan solubilization or AXinsol solubilization or AXinsol degradation.

[00245] In one embodiment, the present invention relates to a method for degrading (or decomposing) insoluble arabinoxylan (AXinsol). It may also be called insoluble arabinoxylan solubilization and/or pentosan solubilization.

[00246] In another embodiment of the present invention, the method is related to the degradation (for example, decomposition) of polymers derived from the degradation of insoluble arabinoxylans.

Arabinoxylan (AX)

[00247] The term "arabinoxylans" (AX), as used herein, means a polysaccharide consisting of a xylan backbone (1,4-linked xylose units) with L-arabinofuranose (L-arabinose in its ring form of 5 atoms) randomly linked by 1α^2 and/or 1α^3 bonds to xylose units throughout the chain. Arabinoxylan is a hemicellulose found in the primary and secondary cell walls of plants. Arabinoxylan can be found in the bran of grains such as wheat, corn, maize, rye and barley.

[00248] Arabinoxylan (AX) is found in close association with the plant cell wall, where it acts as a glue linking various building blocks of the wall and plant cell tissue, giving it structural strength and rigidity.

[00249] The term "pentosan", as used herein, is any of a group of carbohydrates that yield pentoses upon complete hydrolysis.

[00250] Since xylose and arabinose (the constituents of arabinoxylans) are both pentoses, arabinoxylans are generally classified as pentosans.

[00251] AX is the major fraction of non-starch polysaccharide (NSP) in several of the most important feedstocks, including wheat and corn.

[00252] Its abundance, location within plant material and molecular structure mean that AX has a major negative impact on feed digestibility, effectively reducing the nutritional value of the raw materials in which it is present. This makes AX an important anti-nutritional factor, reducing the efficiency of animal production.

[00253] In addition, AX can have a major negative impact when trying to decompose material of plant origin, for example, in processes such as brewing, malting, biofuel manufacturing, effectively reducing the amount of accessible substrate in the raw material of vegetable origin.

[00254] AXs can also hold substantial amounts of water (what might be called their water holding capacity) - this can make arabinoxylans soluble resulting in (high) viscosity - which is a disadvantage in many applications.

[00255] The term "hemicellulose" as used herein means the polysaccharide components of plant cell walls other than cellulose. The term "hemicellulose", as used herein, can mean polysaccharides in plant cell walls that are extractable by dilute alkaline solutions. Hemicelluloses comprise nearly one-third of the carbohydrates in woody plant tissue. The chemical structure of hemicelluloses consists of long chains of a variety of pentoses, hexoses and their corresponding uronic acids. Hemicelluloses can be found in fruits, plant stems and grain husks. Xylan is an example of a pentosan consisting of D-xylose units with 1β^4 linkages.

Water insoluble Arabinoxylan (AXinsol)

[00256] Water-insoluble arabinoxylan (AXinsol), also known as water-inextractable arabinoxylan (WU-AX), constitutes a significant proportion of the dry matter of material of plant origin.

[00257] In wheat, AXinsol may correspond to 6.3% of dry matter. In wheat bran and wheat DDGS, AXinsol can account for about 20.8% or 13.4% of the dry matter (by weight).

[00258] In barley, AXinsol may correspond to 5.5% of the dry matter.

[00259] In maize, AXinsol may correspond to 3.5 to 6% (for example, 5.1%) of dry matter. In maize, AXinsol DDGS can correspond to 10 to 20% (for example, 12.6%) of the dry matter.

[00260] AXinsol causes nutrient entrapment in the feed. Large amounts of well-digestible nutrients, such as starch and protein, remain enclosed in clumps of cell wall material or attached to the side chains of the AX. These trapped nutrients will not be available for digestion and subsequent absorption in the small intestine.

Water-soluble Arabinoxylan (AXsol)

[00261] Water-soluble arabinoxylan (AXsol), also known as water-extractable arabinoxylan (WE-AX), can cause problems in the production of biofuels, in the production of biochemicals, in the processing of carbohydrates and/or in malting and/ or brewing and/or in feed, as it may cause an increase in viscosity due to AXsol's water-binding ability.

[00262] In feed, AXsol may have an antinutritional effect, particularly in monogastric animals, as it causes a considerable increase in the viscosity of the intestinal contents, caused by the extraordinary water binding capacity of AXsol. The increase in viscosity can affect feed digestion and nutrient use as it can prevent the feed from mixing properly with digestive enzymes and bile salts and/or slow nutrient availability and absorption and/or stimulate fermentation. in the large intestine.

[00263] In wheat, AXsol may correspond to 1.8% of dry matter. In wheat bran and wheat DDGS, AXsol can account for about 1.1% or 4.9% of the dry matter (by weight).

[00264] In rye, AXsol may correspond to 3.4% of the dry matter.

[00265] In barley, AXsol may correspond to 0.4 to 0.8% of the dry matter.

[00266] In maize, AXsol may correspond to 0.1 to 0.4% (for example, 0.1%) of dry matter. In maize, AXinsol DDGS can account for 0.3 to 2.5% (eg 0.4%) of dry matter.

[00267] However, in addition to the amount of AXsol present in the plant source material, when a xylanase solubilizes AXinsol in the plant source material, it can release pentosans and/or oligomers that contribute to the AXsol content of the plant source material.

[00268] A significant advantage of the modified xylanases disclosed herein is that they have the ability to solubilize AXinsol without increasing viscosity. It is believed today that high molecular weight products are not formed.

[00269] A decomposition of AXsol can decrease the viscosity.

[00270] A decomposition of AXsol can release nutrients.

Viscosity

[00271] The present invention can be used to ensure that the viscosity is not increased and/or to reduce the viscosity in any process where the water binding ability of AXsol causes an undesirable increase in viscosity.

[00272] The present invention relates to ensuring that the viscosity is not increased and/or to reducing the viscosity by the decomposition (degradation) of AXsol or by the decomposition (degradation) of the polymers and/or oligomers produced by the solubilization of AXinsol.

[00273] Without wishing to be bound by theory, by being able to effectively (rapidly) decompose (degrade) the solubilized polymers (e.g., oligomers) obtained from the dissolution of AXinsol, an undesirable increase in viscosity can be avoided and/or a reduction in viscosity can be obtained. The term "effectively" as used herein means that the enzyme is able to degrade the polymers (e.g., oligomers) formed by AXinsol solubilization faster than the rate at which the AXinsol is degraded (or solubilized).

[00274] Viscosity reduction has advantages in many applications, as taught here.

[00275] An example of a xylanase used in the bioethanol industry is Xylathin™.

[00276] An example of a xylanase used in the wheat starch and gluten separation industry is Shearzyme™.

[00277] In one embodiment of the present invention, the xylanases taught herein are viscosity reducers.

[00278] Generally speaking, wheat (or other cereal) is first dry milled to separate the bran and germ from the endosperm, which is ground into a flour. This endosperm flour is then further fractionated through a wheat starch separation process into various product streams with different commercial values. The main objective is to produce a refined grade of starch A, which consists of large, lenticular granules of 15 to 40 μm. The second stream of starch B consists of less purified starch granules, which are spherical and small (1 to 10 μm). (C.C. Maningat, P.A. Seib, S.D. Bassi, K.S. Woo, G.D. Lasater, chapter 10 of the book "Starch" (2009) 441-451, Wheat starch: production, properties, modification and uses). Isolated wheat starch forms the starting material for the production of modified starch with food and non-food applications. Vital gluten is the third value-added product in wheat separation processes. The vitality of isolated wheat gluten is determined by its ability to form viscoelastic networks necessary for bread production. Vital gluten encapsulates the carbon dioxide formed in the preparation of the dough during baking and consequently increases the volume of the bread. (Anne van der Borght, Hans Goesaert, Wim S. Veraverbeke, Jan A. Delcour, Journal of Cereal Science 41 (2005) 221-237, Fractionation of wheat and wheat flour into starch and gluten: overview of the main processes and the factors involved). It is therefore often used to enrich bread-making flours to obtain improved bakery products. Other markets for gluten include as an additive in vegetarian, meat, fish or poultry products, including those in the pet food industry; in breakfast cereals; or in soy sauce. Due to its thermoplasticity and good film-forming properties, gluten is also used in non-food markets as adhesives. (L. Day, M.A. Augustin, I.L. Batey, C.W. Wrigley, Trends in Food Science & Technology 17 (2006) 82-90, Wheat-gluten uses and industry needs).

[00279] The synthetic xylanases taught herein can be used to reduce viscosity (or not increase viscosity) in processes for separating cereal flour (e.g. wheat, oat, rye or barley flour) into starch and gluten fractions and to optimize the separation by the degradation of oligosaccharides that delay the agglomeration of the gluten.

[00280] The viscosity of wort and the viscosity of barley porridge and barley malt in brewing and malting can cause significant disadvantages during brewing and/or malting. The present invention relates to reducing the viscosity (or not increasing the viscosity) of wort, barley porridge, barley malt or a combination thereof.

ration or food

[00281] The synthetic xylanase or feed additive composition of the present invention can be used as - or in the preparation of - a feed.

[00282] The term "feed" is used synonymously with "feed for animals".

[00283] Preferably, the arabinoxylan-containing material of the present invention is an animal feed or a constituent of an animal feed or a feed component.

[00284] The feed can be in the form of a solution or as a solid or as a semi-solid, depending on the use and/or mode of application and/or mode of administration.

[00285] When used as, or in the preparation of, a feed, as a functional feed, the enzyme or composition of the present invention can be used in conjunction with one or more of: a nutritionally acceptable vehicle, a nutritionally acceptable diluent, a nutritionally acceptable excipient acceptable, a nutritionally acceptable adjuvant, a nutritionally active ingredient.

[00286] In a preferred embodiment, the enzyme or feed additive composition of the present invention is mixed with a feed component to form a feed.

[00287] The term "feed component" as used herein means all or part of the ration. Feed part may mean one feed constituent or more than one feed constituent, for example 2 or 3 or 4. In one embodiment, the term "feed component" encompasses a premix or constituents of premix.

[00288] Preferably, the feed may be a forage, or a premix thereof, a compound feed or a premix thereof. In one embodiment, the feed additive composition according to the present invention can be mixed with a compound feed, a compound feed component, or a premix of a compound feed, or a forage, forage component, or a premix of a forage.

[00289] The term "fodder", for use in the present invention, means any food that is provided to an animal (instead of the animal having to fetch the food itself). Fodder covers plants that have been cut.

[00290] The term forage includes silage, compacted and pelleted feed, oils and mixed feed, as well as sprouted grains and legumes.

[00291] The fodder can be obtained from one or more of the selected plants: corn (maize maize), alfalfa (lucerne), barley, gherkins, brassica, Chau moellier, cabbage, rapeseed (canola), rutabaga (Swedish turnip), turnip, clover, hybrid clover, red clover, subterranean clover, white clover, fescue, bromus, millet, oats, sorghum, soybeans, trees (sprouts from pruned hay trees), wheat and legumes.

[00292] The term "compound feed" means a commercial feed in the form of a flour, a pellet, oilseeds, cake or a bran. Compound feeds can be mixed from various raw materials and additives. These mixtures are formulated according to the specific requirements of the target animal.

[00293] Compound feeds can be complete feeds that provide all the nutrients needed daily, concentrates that provide a portion of the serving (protein, energy) or supplements that only provide additional micronutrients such as minerals and vitamins.

[00294] The main ingredients used in compound feed are feed grains, which include corn, wheat, canola meal, rapeseed meal, lupine, soybean, sorghum, oats and barley.

[00295] Suitably, a premix, as called herein, may be a composition composed of micro-ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. Premixes are generally suitable compositions for mixing into commercial feeds.

[00296] Any animal feed of the present invention may comprise one or more feed materials selected from the group comprising a) cereals, such as small grains (for example, wheat, barley, rye, oats, triticale and combinations thereof) and/ or large grains such as corn or sorghum; b) cereal by-products, such as corn gluten meal, wet cake (particularly corn-based wet cake), dry distillers grain (DDG) (particularly dry corn based distillers grain (cDDG)), solubles distillers dry grain (DDGS) (particularly corn based distillers dry grain (cDDGS) solubles), wheat bran, wheat raw flour, wheat fine flour, rice bran, rice husks, wheat husks oats, palm kernels, and citrus pulp; c) protein obtained from sources such as soy, sunflower, peanuts, lupine, peas, broad beans, cotton, canola, fish meal, dry plasma protein, meat and bone meal, potato protein, whey, copra, sesame ; d) oils and fats obtained from vegetable and animal sources; e) minerals and vitamins.

[00297] In one embodiment, the animal feed comprises or consists of corn, DDGS (such as cDDGS), wheat, wheat bran or a combination thereof.

[00298] In one embodiment, the feed component can be corn, DDGS (e.g., cDDGS), wheat, wheat bran, or a combination thereof.

[00299] In one embodiment, the animal feed comprises or consists of corn, DDGS (such as cDDGS) or a combination thereof.

[00300] In one embodiment, a feed component can be corn, DDGS (such as cDDGS) or a combination thereof.

[00301] An animal feed of the present invention may contain at least 30%, at least 40%, at least 50% or at least 60%, by weight of corn and soybean meal or corn and whole soybean, or wheat bran or sunflower meal.

[00302] An animal feed of the present invention may contain between about 5 to about 40% corn DDGS. For poultry, animal feed can contain, on average, between about 7 to 15% corn DDGS. For swine (pigs), the animal feed can contain, on average, 5 to 40% corn DDGS.

[00303] An animal feed of the present invention may contain corn as the only grain, and in this case, the animal feed may comprise between about 35% and about 80% corn.

[00304] In animal feed comprising mixed grains, for example, comprising corn and wheat, for example, the animal feed may comprise at least 10% corn.

[00305] Additionally or alternatively, the animal feed of the present invention may comprise at least one high-fiber feed material and/or at least one by-product of the at least one high-fiber feed material to provide a high fiber pet food. Examples of high fiber feed materials include: wheat, barley, rye, oats, cereal by-products such as corn gluten meal, corn gluten feed, wet pie, stills dry grain (DDG), corn solubles distillers dry grain (DDGS), wheat bran, raw wheat bran, fine wheat bran, rice bran, rice husks, oat hulls, palm kernel, and citrus pulp. Some protein sources can also be considered high in fiber: protein obtained from sources such as sunflower, lupine, fava beans and cotton.

[00306] In one embodiment, the animal feed of the present invention comprises at least one high-fiber material and/or at least one by-product of the at least one high-fiber feed material selected from the group consisting of solubles distillers dry grain (DDGS), particularly cDDGS, wet cake, distillers dry grain (DDG), particularly cDDG, wheat bran and wheat, for example.

[00307] In one embodiment, the animal feed of the present invention comprises at least one high-fiber material and/or at least one by-product of the at least one high-fiber feed material selected from the group consisting of solubles distillery dry grain (DDGS), particularly cDDGS, wheat bran and wheat, for example.

[00308] In the present invention, the feed can be one or more of the following: a compound feed and premix, including pellets, oleaginous fruits or cake (for cattle); an agricultural crop or agricultural crop residue: maize, soybean, sorghum, oats, barley, copra, straw, millet, sugar beet residue; Fish's flour; meat and bone meal; molasses; solid residue of oilseeds and press cake; oligosaccharides; conserved forage plants: silage; seaweed; seeds and grains, whole or prepared by crushing, milling, etc.; sprouted grains and vegetables; Yeast extract.

[00309] The term "feed" in the present invention also encompasses, in some embodiments, pet food. A pet food is a plant or animal material intended for consumption by pets, such as dog food or cat food. Pet food, such as dog and cat food, can be in a dry form, such as dog chunks, or in a wet canned form. Cat food can contain the amino acid taurine.

[00310] The term "feed" in the present invention also encompasses, in some embodiments, fish food. A fish food will normally contain the macronutrients, trace elements and vitamins needed to keep captive fish in good health. The fish food may be in the form of a flake, pellet or tablet. Pelleted forms, some of which sink quickly, are often used for larger fish or bottom-feeding species. Some fish feeds also contain additives, such as beta-carotene or sex hormones, to artificially enhance the color of ornamental fish.

[00311] The term "feed" in the present invention encompasses, in some embodiments, bird feed. Bird food includes food that is used in bird feeders and to feed pet birds. Typically, bird feed comprises a variety of seeds, but may also include tallow (beef or lamb fat).

[00312] For use in the present invention, the term "contacted" refers to the indirect or direct application of the enzyme (or composition comprising the enzyme) to the product (eg, feed). Examples of application methods that may be used include, but are not limited to, treating the product in a material comprising the feed additive composition, direct application by mixing the feed additive composition with the product, spraying of the feed additive composition onto the surface of the product or dipping the product into a preparation of the feed additive composition.

[00313] In one embodiment, the feed additive composition of the present invention is preferably mixed with the product (e.g., animal feed). Alternatively, the feed additive composition can be included in the emulsion or raw ingredients of an animal feed.

[00314] For some applications, it is important that the composition is made available on or to the surface of a product to be affected/treated. This allows the composition to confer one or more of the following characteristics: performance benefits.

[00315] The synthetic xylanase (or composition comprising the synthetic xylanase) of the present invention can be applied to intercalate, coat and/or impregnate a product (e.g., an animal feed or raw ingredients of an animal feed) with a controlled amount of said enzyme.

[00316] In a particularly preferred embodiment, the enzyme (or composition comprising the enzyme) of the present invention is homogenized to produce a powder.

[00317] In an alternative preferred embodiment, the enzyme (or composition comprising the enzyme) of the present invention is formulated into granules, as described in WO2007/044968 (called TPT granules) or WO1997/016076 or WO1992/012645 here incorporated by way of reference.

[00318] In another preferred embodiment, when the feed additive composition is formulated into granules, the granules comprise a hydrated barrier salt applied as a coating over the protein core. The advantage of such a salt coating is improved thermal tolerance, improved storage stability and protection against other feed additives that otherwise have adverse effects on the enzyme.

[00319] Preferably, the salt used for the salt coating has a water activity greater than 0.25 or constant humidity greater than 60% at 20°C.

[00320] Preferably, the salt coating comprises Na2SO4.

[00321] The method of preparing an enzyme (or composition comprising the enzyme) of the present invention may also comprise the additional step of pelletizing the powder. The powder can be mixed with other components known in the art. The powder, or mixture comprising the powder, may be forced through a die and the resulting strips cut into suitable pellets of varying length.

[00322] Optionally, the pelletizing step may include a treatment with water vapor, or a conditioning stage, before forming the pellets. The mixture comprising the powder may be placed in a conditioner, for example a mixer with steam injection. The mixture is heated in the conditioner to a specified temperature, such as from 60 to 100°C, typical temperatures would be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time can vary from seconds to minutes and even hours. Like, for example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 1 hour.

[00323] It will be understood that the enzyme (or composition comprising the enzyme) of the present invention is suitable for addition to any suitable feed material.

[00324] It will be understood by one skilled in the art that different animals require different foods, and even the same animal may require different foods, depending on the purpose for which the animal is bred.

[00325] Optionally, the animal feed may also contain additional minerals such as, for example, calcium and/or additional vitamins.

[00326] Preferably, the animal feed is a mixture of soybean meal and corn.

[00327] In one embodiment, preferably the feed is not pet food.

[00328] In another aspect, a method for producing an animal feed is presented. Animal feed is typically produced in feed mills in which the raw materials are first ground to an appropriate particle size and then mixed with suitable additives. Animal feed can then be produced as a porridge or pellets; the latter typically involves a method whereby the temperature is raised to a target level and then the feed is passed through a die to produce pellets of a specific size. The pellets are cooled naturally. Subsequently, liquid additives such as fat and enzyme can be added. The production of animal feed may also involve an additional step which includes extrusion or expansion prior to pelletizing, in particular by suitable techniques which may at least include the use of water vapour.

[00329] The animal feed may be an animal feed for a monogastric animal, such as poultry (eg broilers, laying birds, turkey, duck, goose, waterfowl), and swine (all age categories ), a ruminant such as livestock (e.g. cows or oxen (including calves)), horses, sheep, a pet (e.g. dogs, cats) or fish (e.g. agastric fish, gastric fish, water fish sweet like salmon, cod, trout and carp, for example koi carp, marine fish like whiting, and crustaceans like prawns, mussels and scallops). Preferably, the animal feed is for poultry.

Corn-based pet food

[00330] In a preferred embodiment, the animal feed may be a corn-based animal feed. The term "corn-based feed", as used herein, means a feed comprising or consisting of corn (maize) or a by-product of corn.

[00331] Preferably, the corn-based feed comprises corn or a by-product of corn as the main constituent. For example, the corn-based feed may comprise at least 35% corn or a corn by-product, such as at least 40% corn or a corn by-product, such as at least 50% corn or a corn by-product , such as at least 60% corn or a corn by-product, such as at least 70% corn or a corn by-product, such as at least 80% or a corn by-product, such as at least 90% corn or a corn by-product , for example, 100% corn or a by-product of corn.

[00332] In some embodiments, the corn-based feed may comprise corn or a by-product of corn as a minor constituent; and in that case, the animal feed may be supplemented with corn or a by-product of corn. By way of example only, the animal feed may comprise, for example, wheat supplemented with corn or a by-product of corn.

[00333] When corn or corn by-product is a minor constituent of the animal feed, corn or corn by-product makes up at least 5%, preferably at least 10%, preferably at least 20%, preferably , at least 30% of animal feed.

[00334] For clarification, the term "corn" as used herein is synonymous with maize, eg Zea mays.

[00335] In one embodiment, the corn by-product may be corn distillery dry grain (cDDGS) or corn wet cake or corn distillery dry grain (DDG) solubles or corn gluten meal or corn gluten feed corn or combinations thereof.

[00336] In one embodiment, preferably, the arabinoxylan-containing material of the present invention comprises a corn by-product, such as corn distillery dry grain (cDDGS) solubles or corn wet cake or corn distillery dry grain (DDG) solubles or corn gluten meal or corn gluten meal or combinations thereof.

Wheat-based pet food

[00337] In a preferred embodiment, the animal feed may be a wheat-based animal feed. The term "wheat-based animal feed", as used herein, means an animal feed comprising or consisting of wheat or a wheat by-product.

[00338] Preferably, the wheat-based animal feed comprises wheat or a by-product of wheat as the main constituent. For example, the wheat-based animal feed may comprise at least 40% wheat or a wheat by-product, such as at least 60% wheat or a wheat by-product, such as at least 80% wheat or a wheat by-product , such as at least 90% wheat or a wheat by-product, for example, 100% wheat or a wheat by-product.

[00339] In some embodiments, the wheat-based feed may comprise wheat or a wheat by-product as a minor constituent; and in that case, the animal feed may be supplemented with wheat or a wheat by-product. By way of example only, the animal feed may comprise, for example, wheat supplemented with wheat or a wheat by-product.

[00340] When wheat or wheat by-product is a minor constituent of the animal feed, wheat or wheat by-product makes up at least 5%, preferably at least 10%, preferably at least 20%, preferably , at least 30% of animal feed.

[00341] In one embodiment, the wheat by-product can be wheat bran, raw wheat bran, wheat fibers, for example.

[00342] The bran is the hard outer layer of the grain and consists of the combined aleurone and pericarp. Along with the germ, it is an integral part of whole grains, and is often produced as a by-product of milling in the production of refined grains. When the bran is removed from the grains, the grains lose a portion of their nutritional value. Bran is present in any cereal grain, including rice, corn (maize), wheat, oats, barley and millet and can be ground from them. Bran is particularly rich in dietary fiber and essential fatty acids, and contains significant amounts of starch, protein, dietary vitamins and minerals.

[00343] Raw wheat bran is coarse and fine wheat bran particles and fine wheat bran fine particles, wheat germ, wheat flour and offal from the "tail of the mill".

[00344] Raw wheat bran is an inexpensive intermediate by-product of human food and animal feed. In one embodiment, preferably the arabinoxylan-containing material of the present invention comprises wheat bran and/or crude wheat bran.

Wet Cake, Distillery Dry Grain (DDG) and Distillery Dry Grain Solubles (DDGS)

[00345] The wet cake, the dry distillers grains and the dry distillers grains with solubles are products obtained after the removal of ethyl alcohol by distillation from yeast fermentation of a grain or a mixture of grains by methods employed in the industry of grain distillery.

[00346] The stillage from distillation (for example, comprising water, grain residues, yeast cells, etc.) is separated into a "solid" part and a liquid part.

[00347] The solid part is called "wet cake" and can be used as animal feed as it is.

[00348] The liquid part is (partially) evaporated into a syrup (soluble).

[00349] When wet cake is dried it is called distillery dry grain (DDG).

[00350] When wet cake is dried together with syrup (solubles), it is called Distillery Dry Grains with Solubles (DDGS).

[00351] The wet cake can be used in operations with dairy products and feedlots of beef cattle.

[00352] Dry DDGS can be used in livestock feed (eg dairy, beef and swine) and poultry feed.

[00353] Corn DDGS is a great source of protein for dairy cows.

corn gluten meal

[00354] In one aspect, the corn by-product may be corn gluten meal (CGM).

[00355] CGM is a powdered by-product of the corn milling industry. CGM is useful, for example, in animal feed. It can be used as an inexpensive protein source for feeds such as pet food, livestock feed, and poultry feed. It is an especially good source of the amino acid cysteine, but it needs to be balanced with other proteins to provide lysine.

Feed additive composition

[00356] The feed additive composition of the present invention and/or the animal feed comprising the same can be used in any suitable form.

[00357] The feed additive composition of the present invention can be used in the form of solid or liquid preparations or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, pellets, tablets, dusts, and granules which may be wettable, spray-dried or freeze-dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.

[00358] In some applications, the feed additive compositions of the present invention can be mixed with the feed or administered in the drinking water.

[00359] In one aspect, the present invention relates to a method of preparing a feed additive composition, comprising mixing a xylanase, as taught herein, with an acceptable feed vehicle, diluent or excipient and (optionally) packaging.

premix

[00360] The animal feed and/or feed additive composition may be combined with at least one mineral and/or at least one vitamin. The compositions thus derived may be called premixes herein.

Malting and brewing

[00361] The synthetic xylanase (or the composition comprising the synthetic xylanase) of the present invention can be used in malting and brewing industry.

[00362] Barley grains contain 1.7 to 4.1% (w/w) of extractable components in water and 3.6 to 6.4% (w/w) of total beta-glucan (Anderson, M.A., Cook, J.A., & Stone, B.A., Journal of the Institute of Brewing, 1978, 84, 233-239; Henry, J., Journal of the Science of Food and Agriculture, 1985, 36, 1243).

[00363] Wheat grains contain 0.1 to 0.8% (w/w) of extractable components in water and 0.6 to 1.4% (w/w) of total beta-glucan (Anderson, M.A. et al. (1978) above).

[00364] The efficient hydrolysis of arabinoxylans (AXsol) and beta-glucan is important because these compounds can be involved in production problems, such as wort viscosity (Ducroo, P. & Frelon, P.G., Proceedings of the European Brewery Convention Congress , Zurich, 1989, 445; Viêtor, R.J. & Voragen, A.G.J., Journal of the Institute of Brewing, 1993, 99, 243) and filtration capacity and haze formation (Coote, N. & Kirsop, B.H. 1976., Journal of the Institute of Brewing, 1976, 82, 34; Izawa, M., Kano, Y. & Kanimura, M. 1991. Proceedings Aviemore Conference on Malting, Brewing and Distilling, 1990, 427).

[00365] The present invention provides a method of hydrolyzing arabinoxylans (for example, AXinsol and AXsol) during malting and brewing, whereby wheat grains, barley grains or a combination thereof, or portions of the grains wheat and/or barley are mixed with the synthetic xylanase of the present invention.

[00366] In one aspect, the present invention can be associated with a food composition that is a beverage, including but not limited to a fermented beverage such as beer and wine, which comprises a synthetic xylanase according to the present invention.

[00367] In another aspect, the present invention may be associated with a food composition that is a beverage, including, but not limited to, a fermented beverage, such as beer and wine, which comprises a synthetic xylanase according to the present invention .

[00368] In the context of the present invention, the term "fermented beverage" means that it comprises any beverage produced by a method comprising a fermentation process, such as microbial fermentation, such as bacterial and/or yeast fermentation.

[00369] In one aspect of the invention, the fermented beverage is beer. The term "beer" is meant to comprise any fermented wort produced by fermenting/brewing a starch-containing vegetable source material. Often, beer is brewed from malt or ancillary compound, or any combination of malt and ancillary compound, such as starch-containing plant material. As used herein, the term "malt" is understood to mean any malted cereal grain such as malted barley or wheat.

[00370] As used herein, the term "auxiliary compound" refers to any starch and/or sugar containing material of plant origin other than malt, such as barley or wheat malt. As examples of auxiliary compounds, mention may be made of such materials as common corn coarse meal, refined corn coarse meal, ground brewer's yeast, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, toasted cereals, cereal flakes, rye, oats, corn (maize and maize), potatoes, tapioca, cassava and syrups such as corn syrup, cane sugar syrup, invert sugar syrup, syrups of barley and/or wheat and the like can be used as a source of starch.

[00371] As used herein, the term "porridge" (e.g., as used herein in connection with malting or brewing) refers to an aqueous slurry of any material of plant origin containing starch and/or sugar, such as coarse flour, for example, comprising crushed barley malt, crushed barley and/or other auxiliary component or a combination thereof, mixed with water to be further separated into wort and grain residue.

[00372] As used herein, the term "wort" refers to the unfermented liquor that drains away after extracting the grain for grinding during mashing.

[00373] In another aspect, the invention relates to a method of preparing a fermented beverage, such as beer, which comprises mixing the synthetic xylanase of the present invention with malt or an auxiliary compound.

[00374] Examples of beers include: a full malt beer, beer fermented according to the "Reinheitsgebot" (Beer Purity Law), ale, IPA, lager, bitter, Happoshu (second beer), third beer, "dry beer ", "near beer", light beer, low alcohol beer, low calorie beer, porter, bock, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like, but also alternative cereal drinks and malt beverages, such as lemon, orange, lime, or berry flavored malt beverages, liqueur-flavored malt beverages, such as vodka, rum, or tequila-flavored malt beverages, or coffee-flavored malt beverages , such as caffeine-flavored malt liquor, and the like.

Decomposition of grain-based material, e.g. for the production of biofuels

[00375] The synthetic enzyme (or composition comprising the synthetic enzyme) of the present invention, or as disclosed herein, can be used to decompose (degrade) AXinsol and AXsol during the processing of grains from, for example, material grain based. The grain-based material can be whole grains (e.g. wheat, barley, rye, triticale or whole corn grains or mixtures thereof) or portions of whole grains or mixtures thereof.

[00376] In one embodiment, the synthetic enzyme (or composition comprising the synthetic enzyme) of the present invention, or as disclosed herein, can be used to decompose (degrade) AXinsol and AXsol into grain-based materials or into whole grains.

[00377] For clarification, whole grains can be mechanically broken.

[00378] Grain-based material can be decomposed or degraded into glucose. Glucose can subsequently be used as a feedstock for any fermentation process, for example for the production of biofuels (eg bioethanol) and/or for the production of biochemicals (eg biobased isoprene).

[00379] The grain-based material can be feedstock for a biofuel production process (eg bioethanol).

[00380] Today, most fuel ethanol is produced from corn kernels (maize), which is milled, treated with amylase enzymes to hydrolyze the starch into sugars, fermented and distilled. While substantial progress has been made in reducing the costs of ethanol production, substantial challenges remain. Improved techniques are still needed to reduce the cost of biofuel feedstocks for ethanol production. For example, in grain-based ethanol production, degradation of arabinoxylans can increase starch accessibility.

[00381] The present invention provides a synthetic xylanase for use in the decomposition of hemicelluloses, for example arabinoxylan, particularly AXinsol and AXsol.

[00382] Just by way of example, in the European fuel alcohol production industry, small grains such as wheat, barley and rye are common raw materials; in the USA, corn is preferably used. Wheat, barley and rye contain, in addition to starch, high levels of non-starch polysaccharide (NSP) polymers, such as cellulose, beta-glucan and hemicellulose.

[00383] The ratio in which the different NSPs are represented differs for each raw material. The table below shows the different amounts of NSPs in wheat, barley and rye compared to some other raw materials. Table 1: Non-starch polysaccharides (NSPs) present in different raw materials (g/kg of dry matter) 1 Non-cellulosic polysaccharides: pentosans, (arabino)xylans and other hemicelluloses

[00384] NSPs can give high viscosity to grain porridges. High viscosity has a negative impact on ethanol production as it limits the concentration of solids that can be used in mashing and reduces the energy efficiency of the process. In addition, residual hemicelluloses present throughout the process can contribute to fouling in heat exchangers and distillation equipment. The greatest impact of a high viscosity is seen when a porridge is cooled down to fermentation temperature (32°C). This explains why the viscosity needs to be reduced in the process any time before the cooling step.

[00385] In one embodiment of the present invention, the method for degrading the grain-based material comprises mixing the synthetic xylanase, disclosed herein, as early as possible in the process of producing biofuels (e.g., bioethanol), for example, from preferably during the mixing of the grain-based material at the beginning of the process. An advantage of adding the modified xylanases disclosed here at an early stage in the process is that the enzymes reduce the initial viscosity.

[00386] In one embodiment of the present invention, the method for degrading the grain-based material comprises mixing the synthetic xylanase disclosed herein before or during simultaneous liquefaction, saccharification, fermentation, saccharification and fermentation, after fermentation or a combination of the same.

[00387] In one embodiment of the present invention, the method for degrading the grain-based material comprises mixing the synthetic xylanase disclosed herein during liquefaction (e.g., a step at an elevated temperature performed after mixing).

[00388] Therefore, in one embodiment, the present invention relates to the reduction of viscosity when degrading grain-based materials, for example, in biofuel production processes (for example, bioethanol).

[00389] The benefits of using the synthetic xylanases taught here to reduce viscosity when degrading grain-based materials, for example, in biofuel production processes (eg bioethanol) are numerous:

[00390] • A porridge with a higher dry substance content can be used in the process

[00391] • A higher solids content of the final syrup can be obtained

[00392] • Better heat transfer, lower energy requirement

[00393] • Lower evaporator fouling leading to lower cleaning costs

[00394] • Higher final yields of ethanol

[00395] • Improved quality of DDGS (by-product)

[00396] • Better separation between the solid and liquid parts during the separation of vinasse (after distillation). Lower viscosity increases separation efficiency.

[00397] Another significant advantage of the present invention is that the use of the synthetic xylanase described herein in the production of biofuels can also result in improved (by)products of this process, such as wet cake, dry distillery grain (DDG) or dry grain of distillery with solubles (DDGS). Therefore, an advantage of the present invention is that since wet cake, DDG and DDGS are (by)products of the production of biofuels (e.g. bioethanol), the use of the present invention can result in improved quality of these (sub) products. For example, arabinoxylans in (by)products can already be dissolved during the biofuel production process.

Grain gluten-starch separation (e.g. wheat)

[00398] The synthetic xylanase (or composition comprising the synthetic xylanase) of the present invention, or as disclosed herein, can be used to decompose (degrade) AXinsol and AXsol during the separation of starch and gluten from wheat.

[00399] After the initial separation of wheat bran and endosperm germ, the fractionation of wheat endosperm flour into starch and gluten fractions is applied industrially on a large scale to obtain high-quality starch A and by-products starch B and gluten vital.

[00400] The degradation product of cereal flour (eg wheat flour) in the present invention is starch (high quality A starch).

[00401] In addition, B-starch and vital gluten by-products are also produced. Each individual product is then further processed to supplement or modify the characteristics of the food product for market needs.

[00402] There are several wheat separation processes used by the industry described in the literature. These industrial processes differ mainly in the forms of the flour and water mixtures presented to the fractionating equipment (centrifuge, hydrocyclone or screen) or in the initial reaction conditions such as temperature and application of shear (Abdulvahit Sayaslan, Lebensm.-Wiss. U.- Technol 37 (2004) 499-515, Wetmilling of wheat flour: industrial processes and small-sacale test methods).

[00403] In the method for separating a cereal flour (for example, wheat flour) into the starch and gluten fractions, the method comprises mixing a cereal flour (for example, wheat flour), water and a synthetic xylanase. The cereal flour, water and synthetic xylanase can be mixed simultaneously or sequentially. In some embodiments, the cereal flour (e.g., wheat flour) and water can be mixed prior to mixing with the synthetic xylanase.

[00404] In general, cereal flour (for example, wheat flour) is mixed into a dough, ranging from 35 to 63% dry solids, at temperatures of approximately 20 to 45°C. The mixture is then further processed by:

[00405] 1) let the mixture rest for some time (approximately 30 minutes) and then remove the starch by washing the mixture using a screen, centrifuge or hydrocyclone to separate the starch milk from the gluten or

[00406] 2) apply shear to the mixture, optionally diluting the mixture further and then separating the wheat flour by a hydrocyclone or a 2- or 3-phase decanter centrifuge.

[00407] The term "dry solids" as used herein means total solids (dissolved and undissolved) of an aqueous slurry (in %) on a dry weight basis.

[00408] In one embodiment of the present invention, the method or use claimed herein may include the steps of mixing wheat flour to form a dough with between 35 to 63% dry solids, at a temperature of about 20 to about 45 °C and separate the starch from the gluten.

[00409] The method of the present invention may additionally comprise:

[00410] a) let the mixture rest for about 30 minutes and then remove the starch by washing the mixture using a screen, centrifuge or hydrocyclone to separate the starch milk from the gluten or

[00411] b) apply shear to the mixture and, optionally, dilute the mixture further, separating the starch from the gluten using a hydrocyclone, or a 2- or 3-phase decanter centrifuge.

[00412] The present invention provides an improvement in the separation of starch and gluten by the addition of a synthetic xylanase as taught in the present invention, suitably during the initial mixing step of flour and water in the various processes used to separate starch from wheat, described above. Separation is enhanced by the addition of a synthetic xylanase during the initial mixing step due to the reduction in viscosity and hydrolysis of AXsol and/or AXinsol which interfere with the gluten particles. By degrading these poly and oligosaccharides, gluten agglomeration is intensified, improving gluten productivity. (S.A. Frederix, C.M. Courtin, J.A. Delcour, J. Cereal Sci. 40 (2004) 41-49, Substrate selectivity and inhibitor sensitivity affect xylanase functionality in wheat flour gluten-starch separation).

[00413] An advantage of the present invention is that it results in higher yields of starch A and/or better quality gluten (e.g. better quality vital gluten).

[00414] An advantage of the present invention is that it improves the separation of starch and wheat gluten.

[00415] One way to assess gluten quality is by monitoring gluten agglomeration. When a certain amount of friction through kneading or mixing the dough is applied, the gluten particles tend to clump together into larger particles that form a polymeric network called "vital gluten". "Vital gluten" can be added to food products to improve baked goods properties such as dough strength, shelf life and bread volume (L. Day, M.A. Augustin, I.L. Batey and C.W. Wrigley; Wheat-gluten uses and industry needs; Trends in Food Science & Technology 17 (2006) 82-90).

[00416] In the bakery industry, the quality and quantity of gluten in a wheat flour is determined by the standard ICC test n° 155 (AACC 38-12) with the use of Glutomatic. In this device, a dough is formed from wheat flour (10.0 g) mixed with a small amount of 2% NaCl solution (4.2 to 4.8 ml). After 20 seconds of the mixing step, the dough is kneaded continuously while washing for 5 minutes with a 2% NaCl solution at room temperature (~22°C) pumped through the mixing vessel at a flow rate of ~70 ml/ minute. During this washing step, the starch-containing washing water collects and the gluten particles form a gluten ball inside the Glutomatic sieve holder.

[00417] Gluten quality is measured by evaluating gluten agglomeration. This is done by centrifuging the ball of gluten in a special centrifuge containing a small sieve. The gluten particles that pass through this sieve are weighed (small gluten) and the total amount of gluten is weighed. The gluten index is calculated by (total wet gluten - small wet gluten)/total wet gluten. The more gluten agglomeration is improved, the smaller the small gluten fraction will be and the higher the gluten index value will be. A high gluten content with a theoretical maximum of 100% indicates a high quality gluten ball.

[00418] Another value to quantify the amount of gluten is the dry gluten yield (%). This value is calculated by dividing the number of grams of total dry gluten by the total amount of dry flour that was used in the experiment. The more dry gluten that is recovered, the better the separation.

[00419] This industrial test is currently under adaptation to simulate a mass separation process used in industry.

Dosages

[00420] Preferably, the synthetic xylanase is present in the xylan-containing material (e.g., animal feed) in the range of about 500 XU/kg to about 16,000 XU/kg of xylan-containing material (e.g., feed), plus preferably about 750 XU/kg feed to about 8000 XU/kg xylan-containing material (e.g. feed), preferably about 1500 XU/kg feed to about 3000 XU/kg xylan-containing material (e.g. , feed), preferably about 2000 XU/kg feed to about 2500 XU/kg xylan-containing material (e.g. feed), and even more preferably about 1000 XU/kg xylan-containing material (e.g. feed) to about 4,000 XU/kg of xylan-containing material (eg feed).

[00421] In one embodiment, the synthetic xylanase is present in the xylan-containing material (e.g. animal feed) at greater than about 500 XU/kg of xylan-containing material (e.g. feed), suitably more than about 600 XU/kg of xylan-containing material (e.g. feed), suitably more than about 700 XU/kg of xylan-containing material (e.g. feed), suitably more than about 800 XU/kg of xylan-containing material (e.g. , feed), suitably more than about 900 XU/kg xylan-containing material (e.g. feed), suitably more than about 1000 XU/kg xylan-containing material (e.g. feed), suitably more than about 2000 XU/kg, suitably more than about 2500 XU/kg, and suitably more than about 3000 XU/kg of xylan-containing material (eg feed).

[00422] In one embodiment, the synthetic xylanase is present in the xylan-containing material (eg, animal feed) at a concentration of between about 2,000 XU/kg to about 2,500 XU/kg.

[00423] In one embodiment, the synthetic xylanase is present in the xylan-containing material (e.g., animal feed) at less than about 16,000 XU/kg of xylan-containing material (e.g., feed), suitably less than about 8,000 XU/kg of xylan-containing material (e.g. feed), suitably less than about 7000 XU/kg of xylan-containing material (e.g. feed), suitably less than about 6000 XU/kg of xylan-containing material (e.g. , feed), suitably less than about 5000 XU/kg xylan-containing material (e.g. feed), suitably less than about 4000 XU/kg xylan-containing material (e.g. feed).

[00424] Preferably, the synthetic xylanase may be present in a feed additive composition in a range from about 100 XU/g to about 320,000 XU/g of composition, more preferably from about 300 XU/g of composition to about from 160,000 XU/g of composition, and even more preferably, from about 500 XU/g of composition to about 50,000 XU/g of composition, and even more preferably, from about 500 XU/g to about 40,000 XU/g of composition.

[00425] In one embodiment, the synthetic xylanase is present in the feed additive composition at more than about 100 XU/g of the composition, suitably more than about 200 XU/g of the composition, suitably more than about 300 XU/g of the composition g of the composition, suitably more than about 400 XU/g of the composition, and suitably more than about 500 XU/g of the composition.

[00426] In one embodiment, the synthetic xylanase is present in the feed additive composition at less than about 320,000 XU/g of the composition, suitably less than about 160,000 XU/g of the composition, suitably less than about 50,000 XU/g of the composition g of the composition, suitably less than about 40,000 XU/g of the composition, suitably less than about 30,000 XU/g of the composition.

[00427] Xylanase activity can be expressed in xylanase units (XU) measured at pH 5.0 with AZCL-arabinoxylan (azurine cross-linked wheat arabinoxylan, Xylazyme tablets, Megazyme) as substrate. Hydrolysis by endo-(1-4)-β-D-xylanase (xylanase) produces dyed fragments soluble in water, and their release rate (increase in absorbance at 590 nm) can be directly related to enzyme activity. Xylanase Units (XU) are determined relative to a standard enzyme (Danisco xylanase, available from Danisco Animal Nutrition) under standard reaction conditions, which are 40 °C, 5 minute reaction time in McIlvaine buffer, pH 5 ,0.

[00428] The xylanase activity of the standard enzyme is determined as the amount of final reducing sugar groups released from an oat-spelt-xylan substrate per minute at pH 5.3 at 50°C. The final groups of the reducing sugar react with 3,5-dinitrosalicylic acid and the formation of the reaction product can be measured as an increase in absorbance at 540 nm. Enzymatic activity is quantified in relation to a xylose standard curve (reducing sugar equivalents). One xylanase unit (XU) is the amount of standard enzyme that liberates 0.5 μmol of reducing sugar equivalents per minute at pH 5.3 and 50°C.

[00429] In one embodiment, suitably, the enzyme is classified using the EC classification above, and the EC classification designates an enzyme that has this activity when tested in the assay taught here to determine 1 XU.

[00430] Preferably, the synthetic xylanase is present in the mixing step of a wheat starch separation process in the dough or paste in the range of 0.01 kg/MT DS of dough or paste to about 0.60 kg/MT DS, more preferably from about 0.05 kg/MT DS to about 0.45 kg/MT DS of dough or paste, and even more preferably, from about 0.10 kg/MT DS to about 0.25 kg/MT DS of mass or paste.

[00431] In some embodiments (particularly in the wheat starch separation embodiment) the synthetic xylanase can be dosed in the range of about 0.019 g protein/MT DS of wheat flour (which is equivalent to 0.019 mg/kg DS) to about 119 g protein/MT DS of wheat flour (which is equivalent to 119 mg/kg DS - where DS stands for dry solids content and MT stands for metric ton.

[00432] In some embodiments (particularly in the wheat starch separation embodiment), the synthetic xylanase can be dosed at about 1.19 g protein/MT DS of wheat flour (which is equivalent to about 1.19 mg/kg of DS), where DS means dry solids content and MT means metric ton.

[00433] In some embodiments (particularly in the wheat starch separation embodiment), the synthetic xylanase can be dosed in the range of about 9 to about 120,000 units/kg of wheat flour, suitably between about 500 to 2,400 units /kg of wheat flour, suitably between about 900 to 1200 units/kg of wheat flour (where 1 unit is defined as the amount of enzyme required to generate 1 micromol of xylose reducing sugar equivalents per minute under the conditions of the birch wood essay taught below:

birch wood test

[00434] The xylanase activity of an enzyme can be measured using 1% birch wood xylan (Sigma 95588) or wheat flour arabinoxylan (Megazyme P-WAXYM) as substrates. The assay is performed in 50 mM sodium citrate, pH 5.3, with 0.005% Tween-80 buffer at 50 °C for 10 minutes.

[00435] The released reducing sugar is quantified by reaction with 3,5-dinitrosalicylic acid and by measuring the absorbance at 540 nm. Enzymatic activity was quantified in relation to a xylose standard curve. In this assay, one xylanase unit (U) is defined as the amount of enzyme required to generate 1 micromole of xylose reducing sugar equivalents per minute under the assay conditions.

[00436] In some embodiments (particularly in the degradation of grain-based material) the synthetic xylanase can be dosed in the range of about 0.29 g/protein/MT Wheat DS (which is equivalent to 0.29 mg/kg DS) to about 0290 g/protein/MT Wheat DS (which is equivalent to 290 mg/kg DS).

[00437] In some embodiments (particularly grain-based material degradation), xylanase can be administered at 2.9 g/protein/MT Wheat DS (which is equivalent to 2.9 mg/kg DS).

[00438] In some embodiments (particularly in the degradation of grain-based material) xylanase can be dosed in the range of about 22 to about 285,000 units/kg, suitably from about 1,100 to about 5,700 units/kg, suitably from about 2200 to about 2850 units/kg (where 1 unit is defined as the amount of enzyme required to generate 1 micromole of xylose reducing sugar equivalents per minute under the birch wood test conditions discussed above).

[00439] The synthetic enzyme and/or the composition comprising the synthetic enzyme according to the present invention can be designed for a single dose, or can be designed for use (e.g. feed) on a daily basis.

[00440] The ideal amount of the synthetic enzyme and/or the composition comprising the synthetic enzyme to be used in the present invention will depend on the product to be treated and/or the method of contacting the product with the composition and/or the intended use for the same.

[00441] The amount of synthetic enzyme used in the compositions should be a sufficient amount to be effective.

[00442] The amount of synthetic enzyme used in the compositions should be an amount sufficient to be effective and to remain effective enough, for example, in improving the performance of an animal fed feed products containing said composition. This time period for effectiveness should extend to at least the use time of the product (eg feed additive composition or feed containing the same).

Formulation

[00443] In one embodiment, the synthetic enzyme can be formulated as a liquid, a dry powder or a granule.

[00444] The dry powder or granules can be prepared by means known to those skilled in the art, such as in a fluid bed coating application device with upper atomization, in a Wurster with lower atomization, or by drum granulation (e.g. , high shear granulation), extrusion, drum coating or in a micro-ingredient mixer.

[00445] For some embodiments, the synthetic enzyme can be coated, e.g., encapsulated.

[00446] In one embodiment, the coating protects the synthetic xylanase from heat and can be considered a heat protectant.

[00447] In one embodiment, the feed additive composition is formulated as a dry powder or granules as described in WO2007/044968 (called TPT granules) or WO1997/016076 or WO1992/012645 (each of which is incorporated herein , for reference).

[00448] In one embodiment, the feed additive composition can be formulated as a granule for feed compositions comprising: a core; an active agent and at least one coating, the granule active agent retaining at least 50% activity, at least 60% activity, at least 70% activity, at least 80% activity after conditions selected from one or more more of a) a feed pelletizing process, b) a process of pre-treating the heated feed with steam, c) storage, d) storage as an ingredient in an unpelleted mixture, and e) storage as an ingredient in a feed masterbatch or a feed premix comprising at least one compound selected from trace minerals, organic acids, reducing sugars, vitamins, choline chloride and compounds that result in a feed masterbatch or premix of acidic or basic feed.

[00449] With respect to the granule, at least one coating may comprise a moisture hydrating material that constitutes at least 55% w/w of the granule; and/or the at least one coating may comprise two coatings. The two coatings can be a moisture hydrating coating and a moisture barrier coating. In some embodiments, the moisture hydration coating can represent between 25% and 60% w/w of the granule and the moisture barrier coating can represent between 2% and 15% w/w of the granule. The moisture hydrating coating can be selected from inorganic salts, sucrose, starch, and maltodextrin, and the moisture barrier coating can be selected from polymers, gums, whey, and starch.

[00450] The granule can be produced using a feed pelleting process and the feed pretreatment process can be conducted between 70°C and 95°C for up to several minutes, such as between 85°C and 95°C.

[00451] In one embodiment, the feed additive composition can be formulated as an animal feed granule comprising: a core; an active agent, the active agent in the granule retaining at least 80% activity after storage and after a steam heated pelletizing process where the granule is an ingredient; a moisture barrier coating and a hydration coating with moisture that is at least 25% w/w of the granule, the granule having a water activity of less than 0.5 prior to the pelletizing process heated with steam .

[00452] The granule may have a moisture barrier coating selected from polymers and gums, and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating can represent between 25% and 45% w/w of the granule and the moisture barrier coating can represent between 2% and 10% w/w of the granule.

[00453] The granule can be produced using a pelletizing process heated with steam that can be conducted between 85°C and 95°C for up to several minutes.

[00454] In some embodiments, the enzyme can be diluted using a diluent, such as starch powder, limestone or the like.

[00455] In one embodiment, the synthetic enzyme or composition comprising the synthetic enzyme is in a liquid formulation suitable for consumption, preferably such liquid consumption contains one or more of: a buffer, salt, sorbitol and/or glycerol.

[00456] In another embodiment, the synthetic enzyme or composition comprising the synthetic enzyme can be formulated by applying, for example, spraying, the enzyme(s) onto a carrier substrate, such as milled wheat, for example.

[00457] In one embodiment, the synthetic enzyme or composition comprising the synthetic enzyme, according to the present invention, can be formulated as a premix. By way of example only, the premix may comprise one or more feed components, such as one or more minerals and/or one or more vitamins.

[00458] In one embodiment, the synthetic enzyme for use in the present invention is formulated with at least one physiologically acceptable vehicle selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a component of wheat, sucrose , starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulphite, formate and mixtures thereof .

Packaging

[00459] In one embodiment, the synthetic enzyme and/or compositions comprising the same (e.g. feed additive composition) and/or premix and/or feed and/or feed according to the present invention are packed.

[00460] In a preferred embodiment, the feed additive composition and/or the premix and/or the feed or animal feed are packaged in a bag, such as a paper bag.

[00461] In an alternative embodiment, the feed additive composition and/or the premix and/or the feed or animal feed can be sealed in a container. Any suitable container can be used.

Shapes

[00462] The synthetic enzyme or the composition comprising the synthetic enzyme (for example, the feed additive composition) of the present invention, and other components and/or the animal feed comprising the same can be used in any suitable form.

[00463] The synthetic enzyme or the composition comprising the same (for example, the feed additive composition) of the present invention can be used in the form of solid or liquid preparations, or alternatively thereto. Examples of solid preparations include powders, pastes, boluses, capsules, pellets, tablets, pills, capsules, caplets, solutions or suspensions, dusts and granules which may be wettable, spray dried or freeze dried. Examples of liquid preparations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions.

[00464] The composition comprising the synthetic enzyme may contain flavoring or coloring agents, for immediate, delayed, modified, sustained, pulsed or controlled release applications.

[00465] By way of example, if the composition of the present invention is used in a solid form, for example, pelletized form, it may also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, sodium carbonate calcium, dibasic calcium phosphate and glycine; disintegrators such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[00466] Examples of nutritionally acceptable vehicles for use in preparing the forms include, for example, water, saline solutions, alcohol, silicone, waxes, petroleum gel, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose , magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, essential oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[00467] Preferred excipients of the forms include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.

[00468] For aqueous suspensions and/or elixirs, the composition of the present invention can be combined with various sweetening or flavoring agents, dyeing or coloring matter, with emulsifying and/or suspending agents and with diluents such as water, propylene glycol and glycerin and combinations thereof.

Individual

[00469] The term "subject" as used herein means an animal to which a synthetic xylanase according to the present invention will be or has been administered, or a feed additive composition according to the present invention, or a feed for animals comprising said feed additive composition according to the present invention.

[00470] The term "individual" as used herein means an animal.

[00471] In one embodiment, the subject is a mammal, bird, fish or crustacean that includes, for example, livestock or a domesticated animal (for example, a pet).

[00472] In one embodiment, the "individual" is cattle.

[00473] The term "cattle", as used herein, refers to any farmed animal. Preferably, livestock is one or more of ruminants, such as cattle (for example, cows or oxen (including calves)), monogastric animals such as poultry (including broiler chickens, chickens and turkeys), pigs (including piglets), poultry , aquatic animals such as fish, agastric fish, gastric fish, freshwater fish such as salmon, cod, trout and carp, for example Chinese carp, marine fish such as whiting, and crustaceans such as shrimps, molluscs and scallops), horses (including racehorses), sheep (including lambs).

[00474] In another embodiment, the "individual" is a domesticated or pet animal or an animal kept in a zoo.

[00475] The term "domesticated animal or pet or animal kept in a zoo", as used herein, refers to any relevant animal which includes canines (e.g. dogs), felines (e.g. cats), rodents (eg, guinea pigs, rats, mice), poultry, fish (including freshwater and marine fish) and horses.

Performance

[00476] As used herein, "animal performance" may be determined by the feed efficiency and/or weight gain of the animal and/or the feed conversion ratio and/or the digestibility of a nutrient in a feed (e.g. amino acid digestibility) and/or digestible energy or metabolizable energy in a feed and/or by nitrogen retention and/or by the animals' ability to avoid the negative effects of necrotic enteritis and/or by the individual's immune response.

[00477] Preferably, "animal performance" is determined by the feed efficiency and/or weight gain of the animal and/or the feed conversion ratio.

[00478] By "improved animal performance" it is understood that there is increased feed efficiency and/or increased weight gain and/or reduced feed conversion ratio and/or improved digestibility of nutrients or energy in a feed and/or by retention of improved nitrogen and/or by an enhanced immune response in the individual resulting from the use of the feed additive composition of the present invention in the feed as compared to the feed which does not comprise said feed additive composition.

[00479] Preferably, by "improved animal performance" is meant that there is increased feed efficiency and/or increased weight gain and/or reduced feed conversion ratio.

[00480] As used herein, the term "feed efficiency" refers to the amount of weight gain per unit of feed when the animal is fed ad libitum or a specified amount of feed over a period of time.

[00481] By "increased feed efficiency" it is understood that the use of a feed additive composition, according to the present invention, in the feed results in an increased weight gain per unit of feed ingested compared to an animal fed without said feed additive composition.

Feed conversion ratio (fcr)

[00482] As used herein, the term "feed conversion ratio" refers to the amount of feed given to an animal to increase the animal's weight by a specified amount.

[00483] An improved feed conversion ratio means a lower feed conversion ratio.

[00484] By "lower feed conversion ratio" or "enhanced feed conversion ratio" it is understood that the use of a feed additive composition in a feed results in that a smaller amount of feed needs to be given to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount, when the feed does not comprise said feed additive composition.

nutrient digestibility

[00485] Nutrient digestibility, as used herein, means the fraction of a nutrient that disappears from the gastrointestinal tract or from a specified segment of the gastrointestinal tract, eg, the small intestine. Nutrient digestibility can be measured as the difference between what is given to the subject and what comes out in the subject's stool, or between what is given to the subject and what remains in digestion in a specified segment of the gastrointestinal tract, for example , Hi Leo.

[00486] Nutrient digestibility, as used here, can be measured by the difference between the intake of a nutrient and the nutrient excreted through total collection of faeces over a period of time; or with the use of an inert marker that is not absorbed by the animal and allows the researcher to calculate the amount of nutrient that disappeared in the entire gastrointestinal tract or a segment of the gastrointestinal tract. Such an inert marker can be titanium dioxide, chromic oxide or acid-insoluble ash. Digestibility can be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed.

[00487] Nutrient digestibility, as used herein, encompasses starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.

[00488] Energy digestibility, as used herein, means the gross feed energy consumed minus the gross feed energy consumed or the gross feed energy consumed minus the gross energy of digestion material remaining in a specified segment of the gastrointestinal tract of the animal, for example, the ileus. Metabolizable energy as used herein refers to apparent metabolizable energy and means the gross energy of the feed consumed minus the gross energy contained in the faeces, urine and gaseous products of digestion. Energy digestibility and metabolizable energy can be measured as the difference between gross energy intake and gross energy excreted in the faeces or digestion material present in a specified segment of the gastrointestinal tract using the same methods for measuring nutrient digestibility. , with suitable corrections for nitrogen excretion to calculate feed metabolizable energy.

Combination with other components

[00489] The synthetic xylanase of the present invention can be used in combination with other components.

[00490] In one embodiment, the synthetic xylanase of the present invention can be used in combination with a probiotic or a direct-feeding microorganism (DFM), for example, a direct-feeding bacteria.

[00491] The combination of the present invention comprises the synthetic xylanase of the present invention, or a composition comprising the synthetic xylanase, for example, a feed additive composition, and another component which is suitable for human or animal consumption and which is capable of to provide a medical or physiological benefit to the consumer.

[00492] In one embodiment, the "other component" may be one or more additional enzymes (e.g., other feed enzymes or brewing or malting enzymes, or grain processing enzymes or starch and wheat gluten).

[00493] Additional enzymes suitable for use in the present invention may be one or more of the enzymes selected from the group consisting of: endoglucanases (E.C. 3.2.1.4); celiobiohydrolases (E.C. 3.2.1.91), β-glucosidases (E.C. 3.2.1.21), cellulases (E.C. 3.2.1.74), lichenases (E.C. 3.2.1.73), lipases (E.C. 3.1.1.3), lipid acyltransferases (generally classified as E.C. 2.3 .1.x), phospholipases (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), phytases (e.g. 6-phytase (E.C. 3.1.3.26) or a 3-phytase (E.C. 3.1.3.8), amylases, alpha-amylases (E.C. 3.2.1.1), other xylanases (E.C. 3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.2.1.72, E.C. 3.2.1.136), glycoamylases (E.C. 3.2.1.3), hemicells es , proteases (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)), debranching enzyme, cutinases , esterases and/or mannanases (e.g. a β-mannanase (E.C. 3.2.1.78)).

[00494] In one embodiment (particularly, for feed applications), the other component may be one or more of the enzymes selected from the group consisting of an amylase (including a-amylases (E.C. 3.2.1.1), G4-forming amylases ( E.C. 3.2.1.60), β-amylases (E.C. 3.2.1.2) and Y-amylases (E.C. 3.2.1.3) and/or proteases (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)) and/or a phytase (e.g. a 6-phytase (E.C.3.1.3.26) or a 3-phytase (E.C. 3.1. 38)).

[00495] In one embodiment (particularly, for feed applications), the other component can be a combination of an amylase (e.g., a-amylases (E.C. 3.2.1.1) and a protease (e.g., subtilisin (E.C. 3.4. 21.62)).

[00496] In one embodiment (particularly, for feed applications), the other component may be a β-glucanase, for example an endo-1,3(4)-β-glucanase (E.C. 3.2.1.6).

[00497] In one embodiment (particularly for feed applications), the other component may be a phytase for example a 6-phytase (E.C.3.1.3.26) or a 3-phytase (E.C. 3.1.38).

[00498] In one embodiment (particularly, for feed applications), the other component may be a mannanase (e.g., a β-mannanase (E.C. 3.2.1.78)).

[00499] In one embodiment (particularly, for feed applications), the other component can be a lipase (E.C. 3.1.1.3), a lipid acyltransferase (generally classified as E.C. 2.3.1.x) or a phospholipase (E.C. 3.1. 1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), suitably a lipase (E.C. 3.1.1.3).

[00500] In one embodiment (particularly, for feed applications), the other component may be a protease (e.g., subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4 .21.x) or a keratinase (E.C. 3.4.x.x)).

[00501] In one embodiment, the additional component may be a stabilizer or an emulsifier or a binder or vehicle or a diluent or an excipient or a disintegrator.

[00502] The term "stabiliser" as used herein is defined as an ingredient or combination of ingredients that prevents a product (eg a feed product) from changing over time.

[00503] The term "emulsifier" as used herein refers to an ingredient (eg, a feed ingredient) that prevents emulsions from separating. Emulsions are two immiscible substances, one present as a droplet, contained within the other. Emulsions can consist of oil in water, where the droplet or dispersed phase is oil and the continuous phase is water; or water in oil, where water becomes the dispersed phase and the continuous phase is oil. Foams, which are gas-in-liquid, and suspensions, which are solid-in-liquid, can also be stabilized through the use of emulsifiers.

[00504] As used herein, the term "binder" refers to an ingredient (eg, a feed ingredient) that binds the product through a physical or chemical reaction. During "gelling", for example, water is absorbed, providing a binding effect. However, binders can absorb other liquids such as oils, keeping them within the product. In the context of the present invention, binders would typically be used in solid or low moisture products, for example, baking products: pastes, bagels, bread and others. Examples of granulation binders include one or more of: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia.

[00505] The term "vehicle" means materials suitable for administering the enzyme and includes any materials known in the art, such as any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and does not interact with any component of the composition in a harmful way.

[00506] The present invention provides a method for preparing a composition (for example, a feed additive composition) comprising mixing an enzyme of the present invention with at least one physiologically acceptable carrier selected from at least one of maltodextrin, limestone (carbonate calcium), cyclodextrin, wheat or a wheat component, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate , acetate, phosphate, calcium, metabisulphite, formate and mixtures thereof.

[00507] Examples of "excipients" include one or more of: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols.

[00508] Examples of "disintegrators" include one or more of: starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates.

[00509] Examples of "diluents" include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof.

[00510] The other components can be used simultaneously (for example, when they are mixed together or even when they are released by different routes) or sequentially (for example, they can be released by different routes) with the xylanase of the present invention.

[00511] Preferably, when the feed additive composition of the present invention is mixed with other component(s), the DFM remains viable.

[00512] In one embodiment, preferably the feed additive composition according to the present invention does not comprise chromium or organic chromium.

[00513] In one embodiment, preferably the feed additive according to the present invention does not contain glucanase.

[00514] In one embodiment, preferably the feed additive according to the present invention does not contain ascorbic acid.

Isolated

[00515] In one aspect, preferably, the amino acid sequence, or nucleic acid, or enzyme, according to the present invention, is in an isolated form. The term "isolated" means that the sequence or enzyme or nucleic acid is at least substantially free of at least one other component with which the sequence, enzyme or nucleic acid is naturally associated in nature and as found in nature. The sequence, enzyme or nucleic acid of the present invention can be in a form that is substantially free of one or more contaminants that might otherwise be associated with a substance. As such, for example, they may be substantially free of one or more potentially contaminating polypeptides and/or nucleic acid molecules.

Purified

[00516] In one aspect, preferably, the sequence, enzyme or nucleic acid according to the present invention is in a purified form. The term "purified" means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present in a content of at least about 90%, or at least about 95% or at least about 98%, said level being determined on a dry weight/dry weight basis with respect to the total composition. being considered.

Nucleotide sequence

[00517] The scope of the present invention encompasses the nucleotide sequences that encode proteins having the specific properties as defined in the present invention.

[00518] The term "nucleotide sequence", as used herein, refers to an oligonucleotide sequence or polynucleotide sequence, and the variant, homologues, fragments and derivatives of these substances (as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, it may be double-stranded or single-stranded, representing either the sense or antisense strand.

[00519] The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably this means DNA, more preferably cDNA sequence encoding the present invention.

[00520] In one embodiment, the term "nucleotide sequence" means cDNA.

[00521] In a preferred embodiment, the nucleotide sequence when related to and when covered by the scope per se of the present invention does not include the native nucleotide sequence according to the present invention when in its natural environment and when it is linked to its( s) naturally associated sequence(s) that are/are also in their natural environment(s). For ease of reference, this preferred embodiment will be called "non-native nucleotide sequence". In this sense, the term "native nucleotide sequence" means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment. However, the amino acid sequence falling within the scope of the present invention can be isolated and/or purified after expression of a nucleotide sequence in its native organism. Preferably, however, the amino acid sequence falling within the scope of the present invention can be expressed by a nucleotide sequence in its native organism, but where the nucleotide sequence is not under the control of the promoter with which it is naturally associated within that body.

[00522] Typically, the nucleotide sequence covered by the scope of the present invention is prepared using recombinant DNA techniques (ie recombinant DNA). However, in an alternative embodiment of the invention, the nucleotide sequence could be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215- 23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).

Preparing the Nucleotide Sequence

[00523] A nucleotide sequence that encodes either a protein that has the specific properties as defined herein or a protein that is suitable for modification can be identified and/or isolated and/or purified from any cell or organism that produces said protein. Various methods are well known in the art for identifying and/or isolating and/or purifying nucleotide sequences. By way of example, PCR amplification techniques to prepare more than one sequence can be used once a suitable sequence has been identified and/or isolated and/or purified.

[00524] By way of further example, a genomic DNA and/or cDNA library can be constructed using chromosomal DNA or messenger RNA from the organism producing the enzyme. If the amino acid sequence of the enzyme is known, labeled oligonucleotide probes can be synthesized and used to identify enzyme-encoding clones from the genomic library prepared from the organism. Alternatively, a labeled oligonucleotide probe containing sequences homologous to another known enzyme gene could be used to identify enzyme-encoding clones. In this case, less stringent hybridization and wash conditions are used.

[00525] Alternatively, enzyme-encoding clones could be identified by inserting genomic DNA fragments into an expression vector such as a plasmid, transforming enzyme-negative bacteria with the resulting genomic DNA library, and then inoculating the transformed bacteria on agar plates containing a substrate for the enzyme (i.e. arabinoxylan) thus allowing the clones to express the enzyme to be identified.

[00526] In yet another alternative, the nucleotide sequence encoding the enzyme can be prepared synthetically by established standard methods, for example, the phosphoramidite method described by Beucage S.L. et al., (1981) Tetrahedron Letters 22, pages 1859 to 1869, or the method described by Matthes et al., (1984) EMBO J. 3, pages 801 to 805. In the phosphoramidite method, oligonucleotides are synthesized, for example in an automated DNA synthesizer, purified, annealed, ligated and cloned into suitable vectors.

[00527] The nucleotide sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) according with conventional techniques. Each ligated fragment corresponds to various parts of the entire nucleotide sequence. The DNA sequence can also be prepared by polymerase chain reaction (PCR) using specific primers, for example as described in US 4,683,202 or in Saiki R K et al., (Science (1988) 239, 487-491) .

amino acid sequences

[00528] The scope of the present invention also encompasses amino acid sequences of enzymes with the specific properties as defined herein.

[00529] As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme".

[00530] The amino acid sequence can be prepared/isolated from a suitable source, or it can be made synthetically or it can be prepared using recombinant DNA techniques.

[00531] Preferably, the amino acid sequence when relating to and when covered by the scope itself of the present invention is not a native enzyme. In this sense, the term "native enzyme" means an entire enzyme that is in its native environment and when expressed by its native nucleotide sequence.

Sequence identity or sequence homology

[00532] The present invention also encompasses the use of sequences having a degree of sequence identity or sequence homology to the amino acid sequence of a polypeptide having the specific properties defined herein or of any nucleotide sequence encoding such polypeptide (hereinafter hereinafter referred to as the "homologous sequence"). Here, the term "homologue" means an entity having a certain homology to the amino acid sequence in question and the nucleotide sequences in question. Here, the term "homology" can be equated with "identity".

[00533] The amino acid sequence and/or homologous nucleotide sequence must provide and/or encode a polypeptide that retains functional activity and/or enhances enzyme activity.

[00534] In the present context, in some embodiments, a homologous sequence is one that includes an amino acid or a nucleotide sequence that can have at least 97.7% identity, preferably at least 98 or 99% identity with the sequence in question.

[00535] In some embodiments, a homologous sequence is one that includes an amino acid or nucleotide sequence that can have at least 85% identity, preferably at least 90 or 95% identity, with the sequence in question.

[00536] Typically, the homologues will comprise the same active sites, etc., as the individual's amino acid sequence, for example. While homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention, it is preferable to express homology in terms of sequence identity.

[00537] In one embodiment, a homologous sequence is one that includes an amino acid sequence or nucleotide sequence that has one or more additions, deletions, and/or substitutions compared to the sequence in question.

[00538] In the present context, "the subject sequence" refers to the nucleotide or polypeptide sequence/amino acid sequence according to the invention.

[00539] Preferably, % sequence identity with respect to a polypeptide sequence is determined using SEQ ID NO: 1 as the subject sequence in a sequence alignment. In one embodiment, the polypeptide sequence in question is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

[00540] Preferably, % sequence identity with respect to a nucleotide sequence is determined using SEQ ID NO: 2 as the sequence in question in a sequence alignment. In one embodiment, the subject sequence for nucleotide sequences may be selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.

[00541] A "parent nucleic acid" or "amino acid precursor" means a nucleic acid sequence or amino acid sequence encoding or encoding the parent polypeptide, respectively.

[00542] In one embodiment, the present invention refers to a protein whose amino acid sequence is represented in the present invention, or a protein derived from this (parental) protein, by substitution, deletion or addition of one or more amino acids, such as 2 , 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and which has the activity of the parent protein.

[00543] Suitably, the degree of identity with respect to an amino acid sequence is determined over at least 20 contiguous amino acids, preferably at least 30 contiguous amino acids, preferably at least 40 contiguous amino acids, preferably at least 50 contiguous amino acids preferably at least 60 contiguous amino acids preferably at least 100 contiguous amino acids preferably at least 200 contiguous amino acids.

[00544] In one embodiment, the present invention refers to a nucleic acid sequence (or gene) that encodes a protein, whose amino acid sequence is represented in the present invention or that encodes a protein derived from a (parent) protein, by substitution, deletion or addition of one or more amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and which has the activity of the parent protein.

[00545] In the present context, in one embodiment, a homologous sequence, or foreign sequence, is one that includes a nucleotide sequence that can have at least 97.7% identity, preferably at least 98 or 99% identity with a nucleotide sequence encoding a polypeptide of the present invention (the subject sequence).

[00546] In another embodiment, a homologous sequence is one that includes a nucleotide sequence that can have at least 85% identity, preferably at least 90 or 95% identity with a nucleotide sequence that encodes a polypeptide of the present invention (the sequence in question).

[00547] Typically, the homologs will comprise the same sequences that encode the active sites of the sequence in question. While homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention, it is preferable to express homology in terms of sequence identity.

[00548] Homology comparisons can be conducted visually, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the % homology or identity between two or more % sequences.

[00549] The % homology or % identity can be calculated over contiguous sequences, that is, a sequence is aligned with the other sequence, and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, a residue at a time. This is called a gapless or "ungapped" alignment. Typically, such gapless alignments are performed only over a relatively small number of residues.

[00550] Although this is a very simple and consistent method, it does not take into account that, for example, in a pair of sequences that would otherwise be identical, an insertion or deletion will cause the following amino acid residues to be placed out of alignment, thus resulting in a possible large reduction in % homology or % identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[00551] However, these more complex methods designate "gap penalties" for each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting greater relationship between the two sequences compared - will achieve a score greater than one with many gaps. "Affine gap costs" are typically used and charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties produce optimized alignments with fewer than gaps. Most alignment programs allow gap penalties to be modified. However, it is preferable to use the default values when using such software for string comparisons.

[00552] The calculation of % homology or % maximum identity, therefore, first requires producing an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform the sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. 1999 Short Protocols in Molecular Biology, 4th Ed - Cap 18), BLAST 2 (see FEMS Microbiol Lett 1999 174( 2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov), FASTA (Altschul et al. 1990 J. Mol. Biol. 403-410) and AlignX , for example. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (See Ausubel et al. 1999, pages 7-58 to 7-60), as, for example, in the GenomeQuest search engine (www.genomequest.com) .

[00553] Although the final % homology or % identity can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairing comparison. Instead, a scaled similarity score matrix is usually used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM 62 matrix, the default matrix for the BLAST suite of programs. Vector NTI programs typically use either the public default values or a custom symbol comparison table, if provided (see the user manual for further details). For some applications, it is preferable to use the default values for the Vector NTI package.

[00554] Alternatively, the percentage of homologies can be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp), based on an algorithm analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73( 1), 237-244).

[00555] Once the software produces an optimal alignment, it is possible to calculate the % homology, preferably the % identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[00556] Gap penalties should be used to determine identity, so preferably the following parameters are used for sequence pair alignment:

[00557] In one embodiment, CLUSTAL may be used with the gap penalty and gap length defined as above.

[00558] Suitably, the degree of identity with respect to a nucleotide sequence or a protein sequence is determined over at least 20 contiguous nucleotides/amino acids, preferably over at least 30 contiguous nucleotides/amino acids, preferably , over at least 40 contiguous nucleotides/amino acids, preferably over at least 50 contiguous nucleotides/amino acids, preferably over at least 60 contiguous nucleotides/amino acids, preferably over at least 100 nucleotides / contiguous amino acids.

[00559] Suitably, the degree of identity with respect to a nucleotide sequence is determined over at least 100 preferably over at least 200 contiguous nucleotides preferably over at least 300 contiguous nucleotides preferably over at least 400 contiguous nucleotides, preferably over at least 500 contiguous nucleotides, preferably over at least 600 contiguous nucleotides, preferably over at least 700 contiguous nucleotides, preferably over of at least 800 contiguous nucleotides.

[00560] Suitably, the degree of identity with respect to a nucleotide sequence can be determined over the entire sequence taught herein.

[00561] Suitably, the degree of identity with respect to a nucleotide sequence can be determined over the entire sequence taught herein as, for example, SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12. Suitably, the degree of identity with respect to a nucleotide sequence can be determined over the entire sequence taught herein as SEQ ID NO: 2.

[00562] Suitably, the degree of identity with respect to a protein (amino acid) sequence is determined over at least 100 contiguous amino acids, preferably at least 200 contiguous amino acids, preferably at least 300 contiguous amino acids.

[00563] Suitably, the degree of identity to an amino acid or protein sequence can be determined over the entire sequence taught herein.

[00564] Suitably, the degree of identity with respect to an amino acid or protein sequence can be determined over the entire sequence taught herein as the mature sequence, e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. Suitably, the degree of identity to an amino acid or protein sequence can be determined over the entire sequence taught here as SEQ ID NO: 1.

[00565] In the present context, the term "query sequence" means a homologous sequence or foreign sequence, which is aligned with a sequence in question to verify whether it is within the scope of the present invention. Accordingly, such query sequence may, for example, be a prior art sequence or a third party sequence.

[00566] In a preferred embodiment, the sequences are aligned by a global alignment program. The identity is calculated by identifying the number of exact matches identified by the program, divided by the length of the sequence in question.

[00567] In one embodiment, the degree of identity between a query sequence and a subject sequence is determined by 1) aligning the two sequences by any suitable alignment program and using the default scoring matrix and default gap penalty , 2) identifying the number of exact matches, where an exact match is where the alignment program has identified an identical amino acid or nucleotide in the two aligned sequences at a given position in the alignment, and 3) dividing the number of exact matches with the length of the string in question.

[00568] In yet another preferred embodiment, the global alignment program is selected from the group consisting of CLUSTAL and BLAST (preferably BLAST), and the identity is calculated by identifying the number of exact matches identified by the program, divided by the length of the string in question.

[00569] The sequences may also have deletions, insertions or substitutions of amino acid residues resulting in a functionally equivalent substance. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar headgroups that have similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[00570] Conservative substitutions can be made, for example, according to the table below. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other:

[00571] The present invention also encompasses homologous substitution (the terms substitution and replacement are both used in the present invention to mean the exchange of an amino acid residue with an alternative residue) that may occur, i.e., the substitution of a similar for or another, such as basic by basic, acid by acid, polar by polar, etc. Non-homologous substitution can also occur, i.e. from one class of residues to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter called Z), diaminobutyric acid ornithine (hereinafter called B ), ornithine norleucine (hereinafter referred to as O), pyrilalanine, thienylalanine, naphthylalanine and phenylglycine.

[00572] Substitutions can also be made with unnatural amino acids, including alpha* and alpha-disubstituted amino acids*, N-alkyl amino acids*, lactic acid*, halides derived from natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine* , p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L—Y—amino butyric acid*, L-α- isobutyric amino*, L-ε-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline# , L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4- isopropyl)*, L-Tic (1,2,3,4--tetrahydro-isoquinoline-3-carboxyl acid)*, L-diaminopropionic acid# and L-Phe (4-benzyl)*. The notation * was used for the purpose of the above discussion (referring to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative while # which was used to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

[00573] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or βalanine residues. A further form of variation, involving the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues in which the α-carbon substituent group is on the nitrogen atom of the residue rather than the α-carbon. Processes for preparing peptides in peptoid form are known in the art, for example, Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[00574] In one embodiment, the xylanase for use in the present invention may comprise a polypeptide sequence shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 or SEQ ID NO: 11, with a conservative substitution of at least one of the amino acids.

[00575] Accordingly, there may be at least 2 conservative substitutions, such as at least 3 or at least 4 or at least 5.

[00576] Suitably, there may be less than 15 conservative substitutions, for example less than 12, less than 10, less than 8 or less than 5.

[00577] Nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. Several different types of oligonucleotide modifications are known in the art. These include methylphosphonate and phosphorothioate backbones and/or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For purposes of the present invention, it should be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications can be performed to enhance the in vivo activity or lifetime of the nucleotide sequence of the present invention.

[00578] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative, fragment or derivative thereof. If the sequence is complementary to a fragment thereof, then the sequence can be used as a probe to identify similar coding sequences in other organisms, etc.

[00579] Polynucleotides that are not 100% homologous to the sequences of the present invention, but are within the scope of the invention, can be obtained in several ways. Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries made from a variety of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologs and fragments thereof, in general, will be capable of selectively hybridizing with the sequences shown in the sequence listing of the present invention. Such sequences may be obtained by probing cDNA libraries made from or genomic DNA libraries from other animal species, and probing these libraries with probes comprising all or part of any of the sequences in the attached sequence listings, under conditions of average the high stringency. Similar considerations apply for obtaining species homologs and allelic variants of the nucleotide or polypeptide sequences of the invention.

[00580] Strain/species variants and homologues can also be obtained using degenerate PCR that uses primers designed to identify sequences within the variants and homologs that encode conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences of various variants/homologues. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[00581] The primers used in degenerate PCR will contain one or more degenerate positions and will be used under lower stringency conditions than those used for cloning sequences with single sequence primers relative to known sequences.

[00582] Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of characterized sequences. This can be useful where, for example, silent codon changes are needed to optimize codon preferences for a specific host cell in which the polynucleotide sequences are being expressed. Other sequence alterations may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

[00583] The polynucleotides (sequence of nucleotides) of the invention can be used to produce a primer, for example, a PCR primer, a primer for an alternative amplification reaction, a probe, for example, labeled with a descriptor tag by means conventional methods using radioactive or non-radioactive markers, or the polynucleotides can be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein.

[00584] The polynucleotides, such as DNA polynucleotides and probes, according to the invention, can be produced by recombination, synthetically or by any means available to those skilled in the art. They can also be cloned by conventional techniques.

[00585] In general, primers will be produced by synthetic means, involving a stepwise production of the desired nucleic acid sequence, one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

[00586] Longer polynucleotides will generally be produced using recombinant means, for example, using PCR (polymerase chain reaction) cloning techniques. The primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

numbering of amino acids

[00587] In the present invention, a specific numbering of amino acid residue positions in the xylanases used in the present invention can be employed. By aligning the amino acid sequence of a sample of xylanases with the xylanase of the present invention (in particular, SEQ ID NO: 1), it is possible to assign a number to an amino acid residue position in said sample xylanase, which corresponds to the position or amino acid residue numbering of the amino acid sequence shown in SEQ ID NO: 1 of the present invention.

Hybridization

[00588] The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention or sequences that are capable of hybridizing with the sequences of the present invention or with the sequences that are complementary thereto.

[00589] The term "hybridization", as used herein, shall include "the process by which a strand of nucleic acid is joined to a complementary strand through base pairing", as well as the process of amplification as performed by technologies from polymerase chain reaction (PCR).

[00590] The present invention also encompasses the use of nucleotide sequences that are capable of hybridizing with sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof.

[00591] The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridizing to the nucleotide sequences presented herein.

[00592] Preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridizing under stringent conditions (e.g., 50°C and 0.2xSSC {1xSSC = 0.15 M NaCl, Na3 citrate to 0.015 M, pH 7.0}) to the nucleotide sequences presented herein.

[00593] More preferably, the term "variant" encompasses sequences that are complementary to sequences that are capable of hybridizing under conditions of high stringency (e.g., 65°C and 0.1xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0}) to the nucleotide sequences presented herein.

[00594] The present invention also relates to nucleotide sequences that can hybridize with the nucleotide sequences of the present invention (including sequences complementary to those presented herein).

[00595] The present invention also relates to nucleotide sequences that are complementary to sequences that can hybridize with the nucleotide sequences of the present invention (including sequences complementary to those presented herein).

[00596] Preferably, hybridization is analyzed on all of the sequences presented herein.

Enzyme expression

[00597] The nucleotide sequence for use in the present invention can be incorporated into a recombinant replicable vector. The vector can be used to replicate and express the nucleotide sequence in protein/enzyme form in and/or from a compatible host cell.

[00598] Expression can be controlled using control sequences, for example regulatory sequences.

[00599] The protein produced by a recombinant host cell by expression of the nucleotide sequence can be secreted or can be contained within the cell depending on the sequence and/or vector used. The coding sequences can be designed with signal sequences that direct the secretion of the substance coding sequences through a specific prokaryotic or eukaryotic cell membrane.

expression vector

[00600] The term "expression vector" means a construct capable of expression in vivo or in vitro.

[00601] Preferably, the expression vector is incorporated into the genome of a suitable host organism. The term "incorporated" preferably encompasses stable incorporation into the genome.

[00602] The nucleotide sequence of the present invention may be present in a vector in which the nucleotide sequence is functionally linked to regulatory sequences capable of providing expression of the nucleotide sequence by a suitable host organism.

[00603] Vectors for use in the present invention can be transformed into a suitable host cell, as described below, to provide expression of a polypeptide of the present invention.

[00604] The choice of vector, for example a plasmid, cosmid or phage vector will often depend on the host cell into which it is to be introduced.

[00605] The vectors for use in the present invention may contain one or more selectable marker genes, such as a gene that confers antibiotic resistance, for example, resistance to ampicillin, kanamycin, chloramphenicol or tetracycline. Alternatively, selection can be done by cotransformation (as described in WO91/17243).

[00606] The vectors can be used in vitro, for example, for the production of RNA or used to transfect, transform, transduce or infect a host cell.

[00607] Thus, in an additional embodiment, the invention presents a method of making the nucleotide sequences of the present invention by introducing a nucleotide sequence of the present invention into a replicable vector, introducing the vector into a compatible host cell and culturing of the host cell under conditions conducive to vector replication.

[00608] The vector may additionally comprise a nucleotide sequence that allows the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

regulatory sequences

[00609] In some applications, the nucleotide sequence for use in the present invention is operably linked to a regulatory sequence that is capable of providing for expression of the nucleotide sequence, as by the chosen host cell. By way of example, the present invention encompasses a vector comprising the nucleotide sequence of the present invention operably linked to such a regulatory sequence, i.e. the vector is an expression vector.

[00610] The term "functionally linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function as intended. A regulatory sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is obtained under a condition compatible with the control sequences.

[00611] The term "regulatory sequences" includes promoters and enhancers and other signals regulating expression.

[00612] The term "promoter" is used in the normal technical sense, e.g., an RNA polymerase binding site.

[00613] The enhanced expression of the nucleotide sequence encoding the enzyme of the present invention can also be obtained by selecting heterologous regulatory regions, for example, promoter, secretion leader and terminator regions.

[00614] Preferably, the nucleotide sequence according to the present invention is operably linked to at least one promoter.

[00615] Other promoters can still be used to direct the expression of the polypeptide of the present invention.

[00616] Examples of promoters suitable for directing transcription of the nucleotide sequence in a bacterial, fungal or yeast host are well known in the art.

[00617] The promoter may further include elements to ensure or enhance expression in a suitable host. For example, elements can be conserved regions such as Pribnow Box or TATA box.

Constructs

[00618] The term "construct", which is synonymous with terms such as "conjugate", "cassette" and "hybrid", includes a nucleotide sequence for use in accordance with the present invention linked directly or indirectly to a promoter.

[00619] An example of an indirect bond is the provision of a suitable spacer group as an intronic sequence, such as the Sh1 intron or the ADH intron, intermediary of the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in relation to the present invention which includes direct or indirect bonding. In some cases, the terms do not encompass the natural combination of the nucleotide sequence encoding the protein normally associated with the wild-type gene promoter and when they are in their natural environment.

[00620] The construct may also contain or express a marker, which allows selection of the genetic construct.

[00621] For some applications, preferably, the construct of the present invention comprises at least the nucleotide sequence of the present invention operably linked to a promoter.

host cells

[00622] The term "host cell", in relation to the present invention, includes any cell that comprises either a nucleotide sequence or an expression vector, as described above, and that is used in the recombinant production of a protein that has the properties specific as defined in the present invention.

[00623] In one embodiment, the organism is an expression host.

[00624] Thus, another embodiment of the present invention provides host cells transformed or transfected with a nucleotide sequence that expresses the protein of the present invention. The cells will be chosen to be compatible with said vector and may, for example, be prokaryotic (for example bacterial), fungal, or yeast cells.

[00625] Examples of suitable bacterial host organisms are gram-positive or gram-negative bacterial species.

[00626] In one embodiment, the xylanases of the present invention are expressed in the expression host Trichoderma reesei.

[00627] In some embodiments, the expression host for the xylanases of the present invention can be one or more of the following fungal expression hosts: Fusarium spp. (such as Fusarium oxysporum); Aspergillus spp. (such as Aspergillus niger, A. oryzae, A. nidulans, A. awamori) or Trichoderma spp. (like T. reesei).

[00628] In some embodiments, the expression host can be one or more of the following bacterial expression hosts: Streptomyces spp. or Bacillus spp. (for example, Bacillus subtilis or B. licheniformis).

[00629] The use of suitable host cells, such as fungal and yeast host cells, can provide post-translational modifications (eg, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine, or threonine phosphorylation), as may be necessary to confer optimal biological activity in recombinant expression products of the present invention.

Body

[00630] The term "organism" in relation to the present invention includes any organism that could comprise the nucleotide sequence encoding the polypeptide, according to the present invention, and/or the products obtained therefrom, and/or in that a promoter can allow the expression of the nucleotide sequence, according to the present invention, when present in the organism.

[00631] In one embodiment, the organism is an expression host.

[00632] Suitable organisms may include a prokaryote, fungus, yeast or a plant.

[00633] The term "transgenic organism" in relation to the present invention includes any organism that comprises the nucleotide sequence that encodes the polypeptide, according to the present invention, and/or the products obtained therefrom, and/or in that a promoter may allow expression of the nucleotide sequence according to the present invention within the organism. Preferably, the nucleotide sequence is incorporated into the genome of the organism.

[00634] The term "transgenic organism" does not include nucleotide coding sequences native in their natural environment, when they are under the control of their native promoter which is also in their natural environment.

[00635] Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, the nucleotide sequence encoding the polypeptide according to the present invention, constructs according to the present invention, vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention or the products thereof.

[00636] For example, the transgenic organism may also comprise the nucleotide sequence encoding the polypeptide of the present invention under the control of a heterologous promoter.

Transformation of host cells/organism

[00637] As indicated earlier, the host organism can be a prokaryotic or eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli, Streptomyces spp. and Bacillus spp., for example Bacillus subtilis.

[00638] Teachings on transforming prokaryotic hosts are well documented in the art, for example, see Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used, then the nucleotide sequence may need to be suitably modified prior to transformation, such as by intron removal.

[00639] Cells of filamentous fungi can be transformed using various methods known in the art, such as a process involving protoplast formation and protoplast transformation followed by regeneration of the cell wall in a known manner. The use of Aspergillus as a host microorganism is described in EP 0 238 023.

[00640] Transformation of prokaryotes, fungi and yeast is generally well known to the person skilled in the art.

[00641] A host organism can be a fungus, such as mold. Examples of such suitable hosts include any member belonging to the genera Trichoderma (e.g. T. reesei), Thermomyces, Acremonium, Fusarium, Aspergillus, Penicillium, Mucor, Neurospora and the like.

[00642] In one embodiment, the host organism can be a fungus. In a preferred embodiment, the host organism belongs to the genus Trichoderma, eg, T. reesei).

Cultivation and production

[00643] Host cells transformed with the nucleotide sequence of the present invention can be cultured under conditions conducive to the production of the encoded polypeptide and which facilitate the recovery of the polypeptide from the cells and/or culture medium.

[00644] The medium used to cultivate the cells can be any conventional medium suitable for the host cell in question to grow and obtain expression of the polypeptide.

[00645] The protein produced by a recombinant cell can be displayed on the surface of the cell.

[00646] The protein can be secreted from the host cells and can conveniently be taken from the culture medium using well known procedures.

Secretion

[00647] Often, it is desirable that the protein be secreted from the expression host into the culture medium from where the protein can be more easily recovered. According to the present invention, the secretion initiation sequence can be selected based on the desired expression host. Hybrid signal sequences can also be used within the context of the present invention.

large scale application

[00648] In a preferred embodiment of the present invention, the amino acid sequence is used for large-scale applications.

[00649] Preferably, the amino acid sequence is produced in an amount of from 1 g per liter to about 100 g per liter of the total cell culture volume after culturing the host organism.

[00650] Suitably, the amino acid sequence can be produced in an amount from 30 g per liter to about 90 g per liter of the total cell culture volume after culturing the host organism.

General techniques of recombinant dna methodology

[00651] The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of one skilled in the art. These techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, chapters 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and D.M.J. Lilley and J.E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is incorporated herein by reference.

Essay activity test

[00652] The xylanases are tested to measure the xylanase activity in WE-AX (water extractable arabinoxylan) from wheat, in the presence of bovine serum albumin (BSA). The amount of terminal reducing groups increases when xylanases active in soluble arabinoxylans hydrolyze the β1-4 bonds in the substrate. Under conditions of heat and alkalinity, the terminal reducing groups react with the colorless PAHBAH (4-para-hydroxybenzoic acid hydrazide), thereby oxidizing the PAHBAH, and the absorbance is measured at 410 nm (Lever, 1972. Analytical Biochemistry 47 , 273-279).

Buffers and reagents:

[00653] 100 mM sodium acetate (NaAc), pH 5.0, 0.10% BSA: 9.6 g sodium acetate trihydrate is dissolved in 800 ml deionized water and the pH is adjusted to 5 .0 with concentrated acetic acid. Subsequently, deionized water is added to 1000 ml. 1.0 g of BSA (Sigma Aldrich, A7906) is dissolved in 1000 ml of 100 mM NaAc solution, pH 5.0.

[00654] WE-AX, 0.5% arabinoxylan substrate, pH 5.0: 0.5 g of soluble wheat arabinoxylan (43 cSt high viscosity Megazyme, P-WAXYH) are moistened with 5 ml of ethanol at 96% and 95 ml of 100 mM NaAc solution, pH 5.0 are added. The solution is heated under stirring until boiling, and then cooled under stirring to room temperature.

[00655] PAHBAH working solution: Three solutions were prepared: 1) 0.5 M sodium hydroxide (NaOH): 10.0 g sodium hydroxide in 500 ml deionized water; 2) 0.5 M HCl: 20.8 ml 37% HCl in 500 ml deionized water; 3) 5% PAHBAH stock solution: 25.0 g PAHBAH (4-hydroxybenzohydrazide, Sigma H9882) are dissolved in 500 ml of 0.5 M HCl. The solution is protected from light and stored at 4°C . Immediately before use, the PAHBAH stock solution was prepared by diluting it five times with 0.5 M NaOH.

Procedure:

[00656] All dilutions were prepared with a biomechanical dispensing robot (Beckman Coulter, USA), in MTPs (stock plate and test plate: 96-well microplates in clear polystyrene, Corning, catalog no. 9017; plate for PCR: VWR, Eu. catalog no. 211-0297; reading plate: Kisker Biotech, catalog no. G080-F)

[00657] 1.3 μl of enzyme sample (concentration in the range of 40 to 65 μg/ml) is diluted with 147 μl of 0.1% BSA buffer, 100 mM NaAc, pH 5.0, in the assay stock plate.

[00658] 2. 25 μl of sample from the assay stock plate is mixed with 150 μl of WE-AX substrate in the assay plate.

[00659] 3. The test plate is incubated at 30°C and shaken at 1150 rpm for 15 minutes on an iEMS shaker (Thermo Scientific).

[00660] 4. After incubation, 45.4 μl of the test plate reaction mixture is mixed with 135 μl of the PAHBAH working solution on a PCR plate.

[00661] 5. The PCR plate is incubated in a PCR machine (Tetrad 2, peltier thermal cycler, Bio-Rad) at 95°C for 5 minutes and then cooled to 20°C for 10 seconds.

[00662] 6. Sample of 100 μl is transferred to a reading plate and the plate is read at 410 nm in a microplate reader (Molecular Devices).

[00663] The activity of all synthetic xylanases was calculated as the average of the three replicates, minus the blank composed of 100 mM sodium acetate, pH 5.0 and 0.1% BSA buffer, instead of enzyme.

thermostability test Buffers and reagents:

[00664] 1% Tween80: 1 g of Tween80 polysorbate (Sigma P8074) is dissolved with 9 ml of MES buffer, pH 6.0, and then further diluted 10 times with MES buffer, pH 6.0.

[00665] 25 mM MES buffer, pH 6.0, 0.00125% Tween 80: 25 mM MES buffer, pH 6.0: 4.88 g of MES (2-(N-morpholino)-ethanesulfonic acid) are dissolved in 800 ml of deionized water and the pH is adjusted to 6.0 with NaOH. 1.25 ml of 1% Tween80 was added, followed by the addition of deionized water to 1000 ml.

Procedure:

[00666] The thermostability of synthetic xylanase is measured by incubating synthetic xylanases at a concentration of approximately 1 μg/ml of protein (range: 0.8 to 1.3 μg/ml) in 25 mM MES buffer, 0.00125% Tween80, pH 6.0 for 10 minutes at elevated temperature. At the end of the incubation, the residual activity for the heat-treated synthetic xylanases is measured as described in the Activity Assay (steps 2 to 6).

[00667] Residual activity of all synthetic xylanases was calculated as the average of three replicates, minus the blank which includes 25 mM MES buffer, 0.00125% Tween80, pH 6.0, instead of the enzyme. Residual activity is calculated as the ratio of the activity measured for the heat-treated sample to the activity measured for an identical sample that has not been incubated at an elevated temperature.

Pepsin resistance assay

[00668] The ability of synthetic xylanases to resist degradation by pepsin is tested at 40°C in a buffer solution at pH 3.5.

[00669] The ability of synthetic xylanases to resist degradation by pepsin is measured by incubating synthetic xylanases in 100 mM glycine buffer solution, pH 3.5, containing 0.2 g/l of pepsin, for 2 hours at 40° C and shaking at 1150 rpm in an iEMS (Thermo scientific) shaker. At the end of the incubation, the residual activity for the synthetic xylanases is measured as described in the Activity Assay (Steps 2 to 6).

Buffers and reagents:

[00670] 100 mM glycine buffer, pH 3.5: 7.52 g of glycine are dissolved in 800 ml of deionized water and the pH is adjusted to 3.5 with HCl. Subsequently, deionized water is added to 1000 ml. Pepsin 0.2 mg/ml solution: 0.2 g of pepsin (Sigma, P-7000) is dissolved in 1000 ml of 100 mM glycine buffer, pH 3.5.

[00671] The residual activity of all synthetic xylanases is calculated as the average of the three replicates, minus the blank, which includes pepsin solution instead of the enzyme. The residual activity of each synthetic xylanase is calculated as the ratio of the activity measured for the pepsin-treated samples to the activity measured in an activity assay that used untreated samples.

solubilization test Buffers and reagents:

[00672] 100 mM MES buffer, pH 6.0: 19.52 g of MES (2-(N-morpholino)-ethanesulfonic acid) are dissolved in 800 ml of deionized water and the pH is adjusted to 6.0 with NaOH . Subsequently, deionized water is added to 1000 ml.

[00673] 10% corn DDGS substrate solution: cDDGS with particle size <212 μm is hydrated in 100 mM MES buffer solution, pH 6.0, by stirring for 15 min at 600 rpm. Immediately after the end of stirring, the pH is adjusted if a reduction in pH was caused by the acidic residues in the cDDGS. 190 µl/well of cDDGS substrate is transferred to substrate plates, which are stored at -20°C until used.

Procedure:

[00674] All dilutions were prepared with a biomechanical dispensing robot (Beckman Coulter, USA), in MTPs (substrate plate and collection plate: 96-well microplates in clear polystyrene, Corning, catalog no. 9017; Filtration: 0.2 µm Filter Membrane, Corning, Catalog No. 3504, Low Profile Microplate: 1.2 ml Square Low Profile Storage Plate, Catalog No. AB-1127, Thermo Scientific.

[00675] 1. 10 μl of enzyme sample (apparent concentration of 150 μg/ml) is added to previously prepared substrate plates.

[00676] 2. Incubation in iEMS at 40 °C for 240 minutes.

[00677] 3. 170 μl sample from the incubated substrate plate is transferred to a filtration plate.

[00678] 4. The filtration plates are placed on collection plates and centrifuged for 10 min at 1666xg.

[00679] 5. Collection plates are stored at -20°C before further analysis.

[00680] 6. 100 μl of the collection plate were diluted with 900 μl of Milli-Q water in a low-profile microplate and mixed for 2 min on a shaker table before being transferred to the Skalar analyzer apparatus.

Quantification of pentosans

[00681] The total amount of C5 sugars (pentosans) in solution is measured by using a continuous flow analyzer (SKALAR system) according to the method described by Rouau and Surget (1994, Carbohydrate Polymers, 24, 123-132) . The supernatants are treated with a mixture of CH3COOH and HCl to hydrolyze the polysaccharides to monosaccharides. Phloroglucinol (1, 3, 5-trihydroxybenzene) was added to react with the monopentoses and monohexoses, forming a colored complex. By measuring absorbance at 550 nm, with 510 nm as the reference wavelength, the pentose concentration in solution was calculated using a xylose standard curve (50 to 400 μg xylose/ml). Unlike pentose-floroglucinol complexes, the absorbance of the hexose-floroglucinol complex is constant at these wavelengths. Glucose (0.3%) was added to the phloroglucinol solution to create a constant glucose signal and to further ensure that there is no interference from the hexose sugars.

[00682] The results can be presented as performance indexes (PI) which were calculated as the ratio between the values after incubation of cDDGS respectively with and without addition of synthetic xylanase: (total amount of C5 sugars in solution after incubation with synthetic xylanase)/(total amount of C5 sugars in solution after incubation without the presence of enzyme).

Pelleting process and determination of residual xylanase activity after a feed containing the polypeptide has been conditioned for 30 seconds at 90°C, for example as part of a pelleting process

[00683] During the pelletizing process, an enzyme can be formulated in a substrate, for example, wheat, and can be formulated in a premix, for example, a corn/soy mixture (such as 61.1% corn , 31.52% Hipro Soya 48 Soybean, 4.00% Soybean Oil, 0.40% Sodium Bicarbonate, 0.25% Leghennen Vitamins/Minerals, 0.20% DL Methionine, 1.46 % dicalcium phosphate, 1.16% limestone).

[00684] Xylanase can be included at a level that ensures that a final target dosage of, for example, 20,000 XU/kg feed is achieved. The premix can be prepared by mixing the formulated enzyme(s) into the substrate, e.g. wheat, forming a feed (flour) mix, e.g. 10 kg corn/soybean feed mix, and mixing for a specified time of, for example, 10 minutes.

[00685] The premix can be added to the feed, for example, 110 kg of feed, and mixed for a specific time, for example, 10 minutes, before conditioning. The feed comprising the enzyme is typically conditioned for 30 seconds at 90°C prior to pelleting.

[00686] The term "conditioning" or conditioning" as used herein means mixing the feed/enzyme mixture and treating it with dry steam to reach a target temperature of 90°C after 30 seconds.

[00687] Conditioning can be performed by placing the feed/enzyme mixture in a mixer, for example, a continuous mixer (such as the KAHL mixer, length 130 cm, diameter 30 cm, speed 155 rpm).

[00688] The dwell time for 300 kg/h is approximately 30 seconds, calculated as follows:

[00689] Capacity: 300 kg/h - 83.3 g/sec.

[00690] Load measured in the mixer: 2,500 g.

[00691] Residence time in the mixer: 2,500 g: 83.3 g/sec. = 30 sec.

[00692] Mounted on the side of the continuous mixer there can be a distributor with a water outlet and 3 steam valves through which the steam can be directed to the flour (eg feed mixture) or feed/enzyme mixture.

[00693] The steam in this system can be supplied by a high-pressure boiler, for example, a Dan Stoker boiler, with a maximum capacity of 400 kg of steam/hour. The tests can be carried out with an overpressurization of 2 atmospheres and the steam can be conducted through a pressure reduction valve, which controls the addition of steam to the continuous mixer. Three manifold valves can be used to fine-tune the desired temperature for the meal (eg feed mix) or feed/enzyme mix. By adding 1% steam the temperature of the meal (eg feed mix) or feed/enzyme mix increases by 14°C.

[00694] After conditioning, the feed/enzyme mixture can be formatted as pellets. Pellets can be formed on a Simon Heesen Pellet Press with a die of 0 3 mm * 35 mm. The capacity is fixed at 300 kg/h and is adjusted for the dosing screw. The flour/premix is heated to the target temperature between 65 and 95°C by steam in the continuous mixer. The amount of steam can be regulated by a pressure reducing valve and a distributor. For each temperature level, a first sample is taken when the operation stabilizes, after 8 to 10 minutes of pelletizing.

[00695] The pelletizing press can be a Simon Heesen Monoroll Labor type with a 7.5 kW motor. Die inner diameter: 173 mm, roller height: 50 mm, roller diameter: 140 mm. Pelletizing press: 500 rpm and nominal capacity: 300 kg/h.

[00696] Samples can be taken after the pelletizing press. They are cooled, for example, in a compartmentalized cooling box with a perforated bottom, Ventilation: 1500 m3 air/h.

[00697] The mixture of feed (flour) containing xylanase and the resulting feed pellets are ground using a Perten laboratory mill, before the xylanase activity is analyzed in the samples using azurine cross-linked arabinoxylan with wheat as substrate. 5.0 g of the ground sample is mixed with 50 ml of McIlvaine buffer, pH 5.0 and stirred with a magnetic stirrer for 10 minutes. The extract is filtered through a glass fiber filter. 100 µl of the extract is mixed with 400 µl of McIlvaine buffer, pH 5.0 and equilibrated at 50°C for 2 min. A 60 mg tablet of Xylazyme (Megazyme, Ireland) is added to start the reaction and the samples are incubated at 50°C for 60 min before the reaction is stopped by the addition of 5 ml of 2% tris(hydroxymethyl)aminomethane (Sigma, T-1503). The solution is mixed using a vortex, allowed to settle for 5 min and mixed again before being centrifuged at 3500 rpm for 10 minutes. The absorbance of the supernatant is measured at 590 nm. Each sample is measured in duplicate.

[00698] The xylanase activity is quantified using a xylanase standard curve prepared with a flour blank (without enzyme) and feed at 90 C. The activity for the flour sample comprising xylanase is defined as 100% and the Residual synthetic xylanase activity in the 90°C conditioned feed pellet is calculated relative to that value.

[00699] The invention will now be described, by way of example only, with reference to the following figures and examples.

Examples Example 1 - Generation of synthetic xylanases Construction of plasmids

[00700] The genes encoding the synthetic xylanases shown in SEQ ID NO: 2, 4, 6, 8, 10 and 12 were generated by means of a de novo synthesis (GeneArt GmbH, Germany). The synthetic xylanases were then cloned by the supplier into the pTTT-pyr2 target vector via a Gateway system recombination technique (Invitrogen, Carlsbad, CA, USA). The resulting expression plasmids pTTTpyr2-synXyn_VAR ( Figure 13 ) expressing the synthetic xylanases were amplified in the Escherichia coli DH5a strain, purified, sequenced, individually ordered into 96 MTPs and used for fungal transformation as described further. The expression vector contains the promoter and terminator regions of T. reesei cbhl allowing a strong inducible expression of a gene of interest, the selective markers amdS from Aspergillus nidulans and pyr2 from T. reesei that confer growth of transformants in minimal medium with acetamide in the absence of uridine. Plasmids are autonomously maintained in the fungal cell due to telomere regions derived from T. reesei. The use of replicative plasmids results in increased frequencies of transformation and circumvents locus-dependent expression problems seen with integrative fungal transformation.

Fungal strains, growth medium and transformation

[00701] The expression plasmids (5 to 10 μl) were transformed using the PEG-protoplast method into a strain of T. reesei with deletion of the most important cellulases and xylanase 2(Δcbh1 Δcbh2 Δegl1 Δegl2 Δegl3 Δegl4 Δegl5 Δegl6 Δbgl1 Δman1 Δxyn2 Prdiv: iRNAxyn1 xyn3: amdS pyr2-). Further down-regulation of background endogenous xylanase 1 and 3 was further achieved by introducing into the genome of the host strain an iRNA interference cassette in order to turn off the expression of xyn1 and xyn3 simultaneously. All high-throughput transformations were performed robotically in a 24-well MTP using Biomek robots (Beckman Coulter, USA). Plasmids with synthetic xylanases were received from suppliers in 96-well microtiter plates arranged according to a predetermined layout. Transformation mixtures containing approximately 1 μg of DNA and 5x 106 protoplasts in a total volume of 50 μl were treated with 200 μl of 25% PEG solution, diluted with 1 volume of a 1.2M sorbitol/Tris a solution. 10 mM, pH7.5/10 mM CaCl2, robotically rearranged into 24-well microtiter plates and mixed with 1 ml of 3% agarose minimal medium containing 1M sorbitol and 10 mM NH4Cl. After growth of transformants, spores from each well were pooled and inoculated into fresh 24-well MTP containing acetamide with additional selective pressure. Once sporulated, spores were collected and used for inoculation of liquid cultures either in a 24-well MTP format or in shake flasks in the following production medium: 37 g/l glucose, 1 g/l sophorose, 9 g/l of amino acids, 10 g/l of (NH4)2SO4, 5 g/l of KH2PO4, 1 g/l of CaCl2x2H2O, 1 g/l of MgSO4x7H2O, 33 g/l of 1,4-Piperazinobis(acid propanesulfonic acid), pH 5.5, 2.5 ml/l 400X T. reesei trace elements (175 g/l citric acid, 200 g/l FeSO4x7H2O, 16 g/l ZnSO4x7H2O, 3.2 g/l 1 of CuSO4x5H2O, 1.4 g/l of MnSO4xH2O, 0.8 g/l of boric acid). 1 ml of production medium was added to produce the synthetic xylanases in 24-well plates. For shake flasks, the volumes were increased.

[00702] The plates were cultured for 6 days at 28°C and 80% humidity with agitation at 200 rpm. Cells were collected by centrifugation at 2500 rpm for 10 minutes and filtered through a Millipore Multiscreen filter plate using a Millipore vacuum system. Culture supernatants were collected by vacuum filtration and used to test their performance as well as expression level.

[00703] The protein profile of whole broth samples was determined by PAGE electrophoresis on NuPAGE® Novex 10% Bis-Tris gel with MES SDS analysis buffer (Invitrogen, Carlsbad, CA, USA). Polypeptide bands were visualized with SimplyBlue SafeStain (Invitrogen, Carlsbad, CA, USA).

[00704] For larger scale production, the fermentation was conducted in a 6-liter autoclavable continuous stirring reactor. The shake flasks were inoculated with spores and incubated with shaking for 3 days at 28°C in the following shake flask medium: 5 g/l (NH4)2SO4, 4.5 g/l KH2PO4, 1 g/l of MgSO4x7H2O, 14.4 g/l of citric acid x1H2O, 1 g/l of CaCl2x2H2O, 27.5 g/l of glucose, 1 drop of antifoaming agent (EROL DF 6000K). The pH was adjusted with NaOH (2M) to 5.5 and the medium was autoclaved for 20 minutes at 122°C. After cooling, 2.5 ml/l T. reesei 400X trace elements (175 g/l citric acid, 200 g/l FeSO4x7H 2O, 16 g/l ZnSO4x7H 2O, 3.2 g/l of CuSO4x5H 2O, 1.4 g/l of MnSO4xH 2O, 0.8 g/l of boric acid) were added. The cells from the shake flask were used to inoculate a bioreactor containing the following bioreactor medium: 4.7 g/l KH2PO4, 1 g/l MgSO4x7H2O, 4.3 g/l (NH4)2SO4, 45 g/l l of glucose, 0.7 g/l of CaCl2x2H2O, 0.3 g/l of antifoaming agent (EROL DF 6000K), 2.5 ml/l of trace elements of T. reesei 400X (175 g/l of citric acid , 200 g/l FeSO4x7H2O, 16 g/l ZnSO4x7H2O, 3.2 g/l CuSO4x5H2O, 1.4 g/l MnSO4xH2O, 0.8 g/l boric acid). The temperature was controlled at 34°C; the pH was continuously controlled by the addition of 20% ammonium hydroxide. Dissolved oxygen was controlled to a minimum saturation of 40% by varying the agitation speed. The exhaled gas content of carbon dioxide and oxygen were measured. When the initial glucose was depleted, a constant glucose/sophorose feed was started. At the same time, the temperature was reduced to and controlled at 28°C, the pH was increased and controlled at 4.5. Fermentation was stopped after 140 hours. The broth was removed from the tank, and the cells were filtered off. After separating the cells, the filtrate was concentrated by ultrafiltration. Finally, the concentrate was filter sterilized and used for pellet stability studies.

Example 2 - Performance of the synthetic silanase

[00705] In addition to being good in bioefficacy (for example, having a positive effect on animal performance) new xylanase products for commercial use, for example for application in feed, also need to have good product characteristics, including processing stability .

[00706] Synthetic polypeptides with xylanase activity were identified. Thermostability was found in synthetic polypeptides. Synthetic xylanases have been found to have high recovery (residual activity) after being subjected to the pelletizing process. The synthetic xylanases were able to degrade WU-AX (water non-extractable arabinoxylan) from DDGS (eg corn DDGS - and thereby solubilize the corn). Furthermore, synthetic polypeptides have been found to be resistant to degradation by pepsin.

[00707] The purpose of synthetic xylanases in feed is to optimize the energy use of an animal feed by having more insoluble fibers digested and incorporated into the solution, and thus making more nutrients available, thus producing more fermentable non-starch polysaccharides (NSP) .

[00708] In addition to high bioefficacy (for example, having a positive effect on animal performance) the new synthetic xylanases taught in the present invention also have good product characteristics, including thermostability, stability against processing heat (for example, pelletizing) and/or pepsin resistance.

[00709] Synthetic xylanases were tested in a series of tests detailed in the materials and methods section below.

Materials and methods Normalization

[00710] Synthetic xylanases were normalized based on activity. Crude samples were diluted 20-fold and 130-fold, respectively, and activity was measured using the activity assay along with a commercial standard curve for xylanase at the following concentrations: 0, 10, 20, 30, 40, 50, 60 and 70 µg/ml. Samples were diluted in 25 mM sodium acetate, 250 mM NaCl, pH 4.0. Samples with an apparent concentration of less than 1000 µg/ml were quantified using the 20-fold dilution, while the remaining samples were quantified using the 130-fold dilution. Crude synthetic xylanase samples were subsequently diluted to an apparent concentration of 150 µg/ml with 25 mM sodium acetate, 250 mM NaCl, pH 4.0 in a microtiter plate (MTP) (the normalized plate) with a well volume of 210 µl. 53 µl of the normalized sample was diluted to an apparent concentration of 50 µg/ml with 107 µl of 25 mM sodium acetate, 250 mM sodium chloride, pH 4.0.

quantification

[00711] Samples on the normalized plate were quantified using the Criterion Gel system. Laemmli sample buffer (Bio-rad #161-0737) was mixed with dithiothreitol (DTT) (Bio-rad #161-0611) and MilliQ water to a final concentration, after enzyme addition, of 50% in sample buffer Laemmli with 50 mM DTT. 70 μl of Laemmli buffer solution were carefully mixed with 10 μl of sample from the normalized plate in parallel with a standard curve made with purified commercial xylanase diluted with 25 mM sodium acetate, 250 mM sodium chloride, pH 4.0, until the following concentrations: 120, 160, 200, 240, 280 and 320 μg/ml.

[00712] The molecular weight (MW), the number of tryptophans and the ratio between the number of tryptophans and the MW for each of the synthetic xylanases are given in Table 1. The concentration of each xylanase was calculated by correcting the apparent concentration of the commercial xylanase, which was done by multiplying the ratio between the number of tryptophans and the MW of the commercial xylanase divided by the ratio between the number of tryptophans and the MW of the synthetic xylanase. The concentrations of all synthetic xylanase samples in the normalized plate were determined to range from 120 to 200 µg/ml. Table 1 MW and ratio between the number of tryptophans and the MW for each of the synthetic xylanases

activity test

[00713] The synthetic xylanases are tested to measure the xylanase activity in WE-AX (water extractable arabinoxylan) from wheat, in the presence of bovine serum albumin (BSA). The amount of reducing end groups increased when xylanases active in soluble arabinoxylans hydrolyzed β1-4 bonds in the substrate. Under conditions of heat and alkalinity, the terminal reducing groups reacted with the colorless PAHBAH (4-para-hydroxybenzoic acid hydrazide), thereby oxidizing the PAHBAH, and the absorbance was measured at 410 nm (Lever, 1972, Analytical Biochemistry, 47, 273-279).

Buffers and reagents:

[00714] 100 mM sodium acetate (NaAc), pH 5.0, 0.10% BSA: 9.6 g sodium acetate trihydrate was dissolved in 800 ml deionized water and the pH was adjusted to 5 .0 with concentrated acetic acid. Subsequently, deionized water was added to 1000 ml. 1.0 g of BSA (Sigma Aldrich, A7906) was dissolved in 1000 ml of 100 mM NaAc solution, pH 5.0.

[00715] WE-AX, 0.5% arabinoxylan substrate, pH 5.0: 0.5 g of soluble wheat arabinoxylan (43 cSt high viscosity Megazyme, P-WAXYH) were moistened with 5 ml of ethanol at 96% and 100 ml of 100 mM NaAc solution, pH 5.0 was added. The solution was heated under stirring until boiling, and then cooled under stirring to room temperature (RT).

[00716] PAHBAH working solution: Three solutions were prepared: 1) 0.5 M sodium hydroxide (NaOH): 10.0 g sodium hydroxide in 500 ml deionized water; 2) 0.5 M HCl: 20.8 ml 37% HCl in 500 ml deionized water; 3) 5% PAHBAH stock solution: 25.0 g PAHBAH (4-hydroxybenzohydrazide, Sigma H9882) was dissolved in 500 ml of 0.5 M HCl. The solution was protected from light and stored at 4°C . Immediately before use, the PAHBAH stock solution was prepared by diluting it five times with 0.5 M NaOH.

Procedure:

[00717] All dilutions were prepared with a biomechanical dispensing robot (Beckman Coulter, USA), in MTPs (stock plate and test plate: 96-well microplates in clear polystyrene, Corning, catalog no. 9017; plate for PCR: VWR, Eu. catalog no. 211-0297; reading plate: Kisker Biotech, catalog no. G080-F)

[00718] 1.3 μl of enzyme sample (concentration in the range of 40 to 65 μg/ml) was diluted with 147 μl of 0.1% BSA buffer, 100 mM NaAc, pH 5.0, in the assay stock plate.

[00719] 2. 25 μl sample from the assay stock plate was mixed with 150 μl of the WE-AX substrate in the test plate.

[00720] 3. The test plate was incubated at 30°C and shaken at 1150 rpm for 15 minutes on an iEMS shaker (Thermo Scientific).

[00721] 4. After incubation, 45.4 μl of the test plate reaction mixture was mixed with 135 μl of the PAHBAH working solution on a PCR plate.

[00722] 5. The PCR plate was incubated in a PCR machine (Tetrad 2, peltier thermal cycler, Bio-Rad) at 95°C for 5 minutes and then cooled to 20°C for 10 seconds.

[00723] 6. Samples of 100 μl were transferred to a reading plate and the plate was read at 410 nm in a microplate reader (Molecular Devices).

[00724] The activity of all synthetic xylanases was calculated as the average of the three replicates, subtracting the blank composed of 0.1% BSA buffer, 100 mM sodium acetate, pH 5.0, instead of enzyme.

thermostability test Buffers and reagents:

[00725] 1% Tween80: 1 g of Tween80 polysorbate (Sigma P8074) was dissolved with 9 ml of MES buffer, pH 6.0, and then further diluted 10 times with MES buffer, pH 6.0.

[00726] 25 mM MES buffer, pH 6.0, 0.00125% Tween 80: 25 mM MES buffer, pH 6.0: 4.88 g of MES (2-(N-morpholino)-ethanesulfonic acid) were dissolved in 800 ml of deionized water and the pH was adjusted to 6.0 with NaOH. 1.25 ml of 1% Tween80 was added, followed by the addition of deionized water to 1000 ml.

Procedure:

[00727] The thermostability of synthetic xylanase was measured by incubating synthetic xylanases at a concentration of approximately 1 μg/ml of protein (range: 0.8 to 1.3 μg/ml) in 25 mM MES buffer, 0.00125% Tween80, pH 6.0 for 10 minutes at elevated temperature. At the end of the incubation, the residual activity for the heat-treated synthetic xylanases was measured as described in the Activity Assay (Steps 2 to 6).

[00728] Residual activity of each synthetic xylanase was calculated as the average of three replicates, minus the blank which includes 25 mM MES buffer, 0.00125% Tween80, pH 6.0, instead of the enzyme. Residual activity is calculated as the ratio of the activity measured for the heat-treated sample to the activity measured for an identical sample that has not been incubated at an elevated temperature.

Pepsin resistance assay

[00729] The ability of synthetic xylanases to resist degradation by pepsin was tested at 40°C in a buffer solution at pH 3.5.

[00730] The ability of synthetic xylanases to resist degradation by pepsin was measured by incubating synthetic xylanases in 100 mM glycine buffer solution, pH 3.5, containing 0.2 g/l of pepsin, for 2 hours at 40° C and shaking at 1150 rpm in an iEMS (Thermo scientific) shaker. At the end of the incubation, the residual activity for the synthetic xylanases was measured as described in the Activity Assay (Steps 2 to 6).

Buffers and reagents:

[00731] 100 mM glycine buffer, pH 3.5: 7.52 g of glycine was dissolved in 800 ml of deionized water and the pH was adjusted to 3.5 with HCl. Subsequently, deionized water was added to 1000 ml. 0.2 mg/ml pepsin solution: 0.2 g of pepsin (Sigma, P-7000) was dissolved in 1000 ml of 100 mM glycine buffer, pH 3.5.

[00732] The residual activity of each synthetic xylanase was calculated as the average of the three replicates, minus the blank, which includes pepsin solution instead of the enzyme. The residual activity of each synthetic xylanase was calculated as the ratio of the activity measured for the pepsin-treated samples to the activity measured in an activity assay that used untreated samples.

solubilization test Buffers and reagents:

[00733] 100 mM MES buffer, pH 6.0: 19.52 g of MES (2-(N-morpholino)-ethanesulfonic acid) were dissolved in 800 ml of deionized water and the pH was adjusted to 6.0 with NaOH . Subsequently, deionized water was added to 1000 ml.

[00734] 10% corn DDGS substrate solution: cDDGS with particle size <212 μm was hydrated in 100 mM MES buffer solution, pH 6.0, by stirring for 15 min at 600 rpm. Immediately after the end of the stirring, the pH was adjusted because a reduction in pH caused by the acidic residues in the cDDGS occurred. 190 µl/well of cDDGS substrate was transferred to substrate plates, which were stored at -20°C until used.

Procedure:

[00735] All dilutions were prepared with a biomechanical dispensing robot (Beckman Coulter, USA), in MTPs (substrate plate and collection plate: 96-well microplates in clear polystyrene, Corning, catalog no. 9017; Filtration: 0.2 µm Filter Membrane, Corning, Catalog No. 3504, Low Profile Microplate: 1.2 ml Square Low Profile Storage Plate, Catalog No. AB-1127, Thermo Scientific.

[00736] 1. 10 μl of enzyme sample (apparent concentration of 150 μg/ml) was added to previously prepared substrate plates.

[00737] 2. Incubation in iEMS at 40 °C for 240 minutes.

[00738] 3. 170 μl sample from the incubated substrate plate was transferred to a filter plate.

[00739] 4. The filtration plates were placed on collection plates and centrifuged for 10 min at 1666xg.

[00740] 5. Collection plates were stored at -20°C before further analysis.

[00741] 6. 100 μl of the collection plate was diluted with 900 μl of Milli-Q water in a low-profile microplate and mixed for 2 min on a shaker table before being transferred to the Skalar analyzer apparatus.

Quantification of pentosans

[00742] The total amount of C5 sugars (pentosans) in solution was measured using a continuous flow analyzer (SKALAR system) according to the method described by Rouau and Surget (1994, Carbohydrate Polymers, 24, 123-132) . Supernatants were treated with a mixture of CH3COOH and HCl to hydrolyze polysaccharides to monosaccharides. Phloroglucinol (1, 3, 5-trihydroxybenzene) was added to react with the monopentoses and monohexoses, forming a colored complex. By measuring absorbance at 550 nm, with 510 nm as the reference wavelength, the pentose concentration in solution was calculated using a xylose standard curve (50 to 400 μg xylose/ml). Unlike the pentose-floroglucinol complex, the absorbance of the hexose-floroglucinol complex is constant at these wavelengths. Glucose (0.3%) was added to the phloroglucinol solution to create a constant glucose signal and to further ensure that there is no interference from the hexose sugars.

[00743] The results are presented as performance indices (PI) which were calculated as the ratio between the values after incubation of cDDGS respectively with and without addition of synthetic xylanase: (total amount of C5 sugars in solution after incubation with xylanase synthetic)/(total amount of C5 sugars in solution after incubation without enzyme).

Pelleting stability

[00744] Pelleting tests were carried out on a full scale at the Technological Institute, Kolding, Denmark. Each xylanase was formulated in wheat and mixed into a corn/soybean feed mix (61.1% corn, 31.52% Hipro 48 soy, 4.00% soy oil, 0.40% sodium bicarbonate , 0.25% Leghennen vitamins/minerals, 0.20% DL methionine, 1.46% dicalcium phosphate, 1.16% limestone).

[00745] Xylanase was included to reach an ultimate target of 20,000 XU/kg feed. A premix was prepared by mixing the wheat-formulated xylanases into 10 kg of corn/soybean feed mix and blending for 10 minutes. The premix was then added to 110 kg of feed and mixed for 10 minutes before conditioning. The feed comprising the enzyme was conditioned for 30 seconds at 90°C before pelleting. The term "conditioning" as used herein means mixing the feed/enzyme mixture and treating it with dry steam to reach a target temperature of 90°C after 30 seconds.

[00746] Conditioning was performed by placing the feed/enzyme mixture in a continuous mixer, specifically a KAHL mixer, 130 cm long, 30 cm in diameter, speed 155 rpm).

[00747] The dwell time for 300 kg/h is approximately 30 seconds, calculated as follows:

[00748] Capacity: 300 kg/h - 83.3 g/sec.

[00749] Load measured in the mixer: 2,500 g.

[00750] Residence time in the mixer: 2,500 g: 83.3 g/sec. = 30 sec.

[00751] Mounted on the side of the continuous mixer was a distributor with a water outlet and 3 steam valves through which steam was directed to the flour (eg feed mix) or feed/enzyme mix.

[00752] The steam in this system was provided by a Dan Stoker high pressure boiler, with a maximum capacity of 400 kg of steam/hour. The tests were carried out with 2 atmospheres overpressurization and the steam could be conducted through a pressure reduction valve, which controls the addition of steam to the continuous mixer. Three manifold valves can be used to fine-tune the desired temperature for the meal (eg feed mix) or feed/enzyme mix. By adding 1% steam the temperature of the meal (eg feed mix) or feed/enzyme mix increases by 14°C.

[00753] After conditioning, the feed/enzyme mixture was formatted as pellets. Pellets were formed on a Simon Heesen pellet press with a 0.3 mm * 35 mm die. The capacity was fixed at 300 kg/h and was adjusted for the dosing screw. The flour/premix was heated to the target temperature between 65 and 95°C by steam in the continuous mixer. The amount of steam was regulated by a pressure reducing valve and a distributor. For each temperature level, a first sample was taken when the operation stabilized, after 8 to 10 minutes of pelletizing.

[00754] The pelletizing press was a Simon Heesen Monoroll Labor type with a 7.5 kW engine. Die inner diameter: 173 mm, roller height: 50 mm, roller diameter: 140 mm. Pelletizing press: 500 rpm and nominal capacity: 300 kg/h.

[00755] Samples were taken after the pelletizing press. They are cooled, for example, in a compartmentalized cooling box with a perforated bottom, Ventilation: 1500 m3 air/h.

[00756] The mixture of feed (flour) containing xylanase and the resulting feed pellets were ground using a Perten laboratory mill, before analyzing the xylanase activity in the samples using azurine-crosslinked arabinoxylan with wheat as substrate. 5.0 g of the ground sample was mixed with 50 ml of McIlvaine buffer, pH 5.0 and stirred with a magnetic stirrer for 10 minutes. The extract was filtered through a glass fiber filter. 100 μl of the extract was mixed with 400 μl of McIlvaine buffer, pH 5.0 and equilibrated at 50°C for 2 min. A 60 mg tablet of Xylazyme (Megazyme, Ireland) was added to start the reaction and the samples were incubated at 50°C for 60 min before the reaction was stopped by the addition of 5 ml of Tris (Sigma, T-1503) to 2%. The solution was mixed by vortexing, allowed to stand for 5 minutes and mixed again before being centrifuged at 3500 rpm for 10 minutes. The absorbance of the supernatant was measured at 590 nm. Each sample was measured in duplicate.

[00757] Xylanase activity was quantified using a standard curve prepared with a porridge blank (without enzyme) and feed at 90°C. The formulated SynXyn92 wheat xylanase was extracted for 10 minutes in McIlvaine buffer, pH 5, 0, to obtain a concentration of 160 XU/ml. The extract was filtered through a glass fiber filter and then 0, 200, 400, 600, 800, and 1000 µl of extract were added to 5.0 g samples of ground control porridge and chow at 90°C. Xylanase activity in these standard samples was measured as described in the extract method above.

[00758] The activity for the meal sample comprising xylanase is defined as 100% and the residual activity of the synthetic xylanase in the feed pellet conditioned at 90 °C is calculated with respect to that value.

Results

[00759] The synthetic xylanases taught here are good for in vitro degrading of WU-AX (water-inextractable arabinoxylans) from cDDGS, have adequate specific activities at pH 5, are thermostable and resistant to degradation by pepsin at pH 3.5 ( with a residual activity of at least 70% after 2 hours of incubation). Feed processing stability was determined for the synthetic enzymes identified here as synXyn92 and feed processing stability was considered high (with a residual activity of at least 80% for treatment at 90°C).

[00760] Table 2: The residual activities after 10 minutes of incubation at selected temperatures (61°C, 65°C or 71°C), the residual activities after 2 hours of incubation in the presence of pepsin and the PI for the solubilization of pentosans by synthetic xylanases are listed here. Synthetic xylanases with PI greater than 1.5 were considered capable of degrading cDDGS WU-AX.

[00761] Table 3 shows the identity in a paired alignment expressed as a percentage. The percent identities were calculated using the Indonesia software package, which was also used to prepare the sequence alignments. Percent identity was calculated by dividing the number of identical amino acids by the number of amino acids in the shorter polypeptide.

[00762] Processing stability, in the form of feed pelleting stability, was determined using a standardized procedure at the Danish Technological Institute and is described under pelleting stability in the materials and methods section. Residual activity after pelleting was 84% for SynXyn92. An 80% recovery after pelleting would be considered a full recovery.

[00763] The synthetic sequences herein named SynXyn 92, SynXyn85, SynXyn89, SynXyn72, SynXyn80 and SynXyn93 also gave a positive response when subjected to the Activity Assay. Polypeptides that result in an OD(410 nm)>0.7 when analyzed under the conditions mentioned in the Activity Assay are considered to have xylanase activity.

Example 3 - Reduction of wheat viscosity

[00764] In the European fuel alcohol production industry, small grains such as wheat, barley and rye are common raw materials in contrast to the US where corn is preferably used. These small grains contain, in addition to starch, high levels of non-starch polysaccharide (NSP) polymers, such as cellulose, beta-glucan and hemicellulose.

[00765] The ratio in which the different NSPs are represented differs for each raw material. Table 1 shows the different amounts of NSPs in wheat, barley and rye compared to some other feedstocks. Table 1: Non-starch polysaccharides present in different raw materials (g kg-1 dry matter)1,2 1 Bach Knudsen, KE, 1997. Carbohydrate and lignin contents of plant materials used in animal feeding. animation Feed Sci. Technol., 67(4): 319338 2 Englyst, HN, Anderson, V. and Cummings, JH, 1983. Starch and non-starch polysaccharides in some cereal foods. J. Sci. Food Agric., 34: 1434-1440. 3 Non-cellulosic polysaccharides: pentosans, (arabino)xylans and other hemicelluloses

[00766] NSPs give high viscosity to grain porridges. High viscosity has a negative impact on ethanol production as it limits the concentration of solids that can be used in mashing and reduces the energy efficiency of the process. In addition, residual hemicelluloses present throughout the process can contribute to fouling in heat exchangers and distillation equipment. The greatest impact of a high viscosity is seen when a porridge is cooled down to fermentation temperature (32°C). This explains why the viscosity needs to be reduced in the process any time before the cooling step. Depending on the process used, it is necessary for the enzymes to run at 60°C and/or 85°C.

[00767] Viscosity-reducing enzymes can be added at different stages of the ethanol production process: blending and/or saccharification/fermentation. Preferably, the enzymes are added in admixture to reduce the initial viscosity.

[00768] The benefits of using viscosity-reducing enzymes in the ethanol production process are several:

[00769] • A porridge with a higher dry substance content can be used in the process

[00770] • A higher solids content of the final syrup can be obtained

[00771] • Better heat transfer, lower energy requirement

[00772] • Lower evaporator fouling leading to lower cleaning costs

[00773] • Higher final yields of ethanol

[00774] • Improved DDGS quality

Methods

[00775] A Perten Instruments Rapid Viscosity Analyzer (RVA 4500) was used to measure the viscosity profiles of a wheat porridge. This wheat porridge was prepared according to the following protocol:

[00776] Prepare 60 grams of aqueous wheat paste at 30% DS (34.65% 'as it is') (for simultaneous testing on two RVA's) as follows:

[00777] - Weigh 20.80 grams of wheat

[00778] - In a 100 ml glass beaker, weigh 39.20 grams of running water and add 137 μl of 4N H2SO4

[00779] - Add the wheat to the water and shake for 5 minutes at maximum speed (approximately 500 rpm) with an overhead shaker

[00780] - Transfer 25.0 grams of aqueous paste to an RVA container, add enzymes diluted 50 times and start the RVA test (check that the initial pH is around 5.3)

[00781] - Check the pH at the end of the RVA test (5.6 to 5.7)

[00782] In each experiment (25 grams of aqueous paste), xylanase was administered to 25 μg of protein (per 8.66 g of wheat 'as it is'), corresponding to 2.9 μg of protein / g of wheat 'as it is'. SPEZYME® CL was administered at 0.15 kg/MT wheat 'as is' (2.2 AAU/g 'as is' or 2.6 AAU/g DS).

[00783] A standard wheat liquefaction was imitated in RVA. The pretreatment was carried out for 20 minutes at 60°C, followed by a liquefaction step for 30 minutes at 85°C. After pre-treatment and liquefaction, the aqueous slurry was cooled to 32°C to determine the viscosity under fermentation conditions. The pH of the liquefaction was maintained between 5.3 to 5.7.

[00784] In this experiment, the performance of SynXyn93 was compared with a known xylanase with viscosity-reducing properties (positive control). The data is shown in Figure 16.

[00785] These data show that the SynXyn93 enzyme has no activity during the pretreatment step (60°C), but recovers in the liquefaction step (85°C). The viscosity reduction continues during the liquefaction step at 85°C, indicating that SynXyn93 has significant activity at this elevated temperature. Final viscosities with SynXyn93 are 52% to 55% lower than the blank.

[00786] A follow-up experiment was carried out by adding the xylanase enzyme at the liquefaction temperature (85°C), instead of adding it at the beginning of the process. This was done to demonstrate the SynXyn93's improved thermostability. In these operations, a rotational axis with an injection opening was used, allowing the addition of enzyme during the operation of the RVA analyzer. The spindle was stopped for 1 minute when the liquefaction temperature (85°C) was reached (at 22 minutes of processing time) and the xylanase enzyme was added. The injection opening was washed with 50 μl of demineralized water and the shaft was rotated again at 23 minutes of processing time). In these operations, SPEZYME® CL was added at the beginning of the process. * No xylanase was added in the pretreatment, therefore the same result as the blank is expected

[00787] The data is shown in Figure 17.

[00788] These data confirm the increased thermostability of SynXyn93 compared to the positive control enzyme. SynXyn93 shows a viscosity reduction of 54% to 60% compared to the blank, while for the positive control enzyme the reduction was only 40% to 41%. For SynXyn93, there is no difference in performance when it is added at the beginning of the process or at 85°C. On the other hand, the positive control enzyme suffers a considerable impact when it is added at 85°C, instead of at the beginning of the process: the viscosity reduction (compared to the blank) decreases from 63% to 41%. Therefore, in this case the final viscosity with SynXyn93 is lower than with the positive control enzyme.

[00789] All publications mentioned in the above specification are hereby incorporated by reference. Various modifications and variations of the described methods and system of the present invention will become apparent to those skilled in the art without departing from the spirit and scope of the invention. While the present invention has been described in connection with specific preferred embodiments, it is to be understood that the invention as claimed is not to be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention that are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims (15)

1. Polypeptide having xylanase activity, characterized in that it comprises an amino acid sequence of SEQ ID NO: 1. 2. Polypeptide, according to claim 1, characterized in that it is an endo-1,4-β-xylanase. 3. Polypeptide, according to claim 1, characterized in that it has at least 50% residual activity of xylanase activity after incubation at 65°C for 10 minutes at pH 6 and/or has at least 80% activity residual xylanase activity after incubation at 61°C for 10 minutes at pH 6. 4. Method for producing the polypeptide, as defined in claim 1, characterized in that it comprises (a) cultivating a host cell comprising a polynucleotide of SEQ ID NO 2 or a degenerate enucleotide sequence thereof that encodes SEQ ID NO: 1, under conditions suitable for producing the polypeptide, and (b) recovering the polypeptide. 5. Method according to claim 4, characterized in that the produced polypeptide is isolated and/or purified. 6. Feed or food for animals, characterized by the fact that it comprises a polypeptide, as defined in claim 1. 7. Method for solubilizing arabinoxylan in a material containing xylan, characterized in that it puts the material containing xylan in contact with the polypeptide, as defined in claims 1. 8. Method according to claim 7, characterized in that the arabinoxylan is an insoluble arabinoxylan (AXinsol). 9. Method, according to claim 7, characterized in that the xylan-containing material is selected from one or more of the group consisting of: an animal feed or food, a feed component, a material based on grains; a porridge; a wort; a malt; a malted barley; an auxiliary compound, a barley porridge; and a cereal flour. 10. Method according to claim 9, characterized in that the feed or animal feed or feed component comprises or consists of corn, DDGS, cDDGS, wheat, wheat bran or a combination thereof. 11. Method according to claim 7, characterized in that the polypeptide is used in combination with one or more of the enzymes selected from the group consisting of endoglucanases (E.C. 3.2.1.4); celiobiohydrolases (E.C. 3.2.1.91), β-glucosidases (E.C. 3.2.1.21), cellulases (E.C. 3.2.1.74), lichenases (E.C. 3.2.1.73), lipases (E.C. 3.1.1.3), lipid acyltransferases (generally classified as E.C. 2.3 .1.x), phospholipases (E.C. 3.1.1.4, E.C. 3.1.1.32 or E.C. 3.1.1.5), phytases (e.g. 6-phytase (E.C. 3.1.3.26) or a 3-phytase (E.C. 3.1.3.8), amylases, alpha-amylases (E.C. 3.2.1.1), other xylanases (E.C. 3.2.1.8, E.C. 3.2.1.32, E.C. 3.2.1.37, E.C. 3.2.1.72, E.C. 3.2.1.136), glycoamylases (E.C. 3.2.1.3), hemicells es , proteases (e.g. subtilisin (E.C. 3.4.21.62) or a bacillolysin (E.C. 3.4.24.28) or an alkaline serine protease (E.C. 3.4.21.x) or a keratinase (E.C. 3.4.x.x)), debranching enzyme, cutinases , esterases and/or mannanases (e.g. a β-mannanase (E.C. 3.2.1.78)). 12. Method according to claim 8, characterized in that the polypeptide is used in combination with one or more of the enzymes selected from the group consisting of endoglucanases (EC 3.2.1.4), celiobiohydrolases (EC 3.2.1.91), β-glucosidases (EC 3.2.1.21), cellulases (EC 3.2.1.74), lichenases (EC 3.2.1.73), lipases (EC 3.1.1.3), lipid acyltransferases (EC 2.3.1.x), phospholipases (EC 3.1. 1.4, EC 3.1.1.32 and EC 3.1.1.5), 6-phytase (EC 3.1.3.26), 3-phytase (EC 3.1.3.8), amylases, alpha-amylases (EC 3.2.1.1), xylanases (EC 3.2. 1.8, EC 3.2.1.32, EC 3.2.1.37, EC 3.2.1.72 and EC 3.2.1.136), glucoamylases (EC 3.2.1.3), hemicellulases, subtilisin (EC 3.4.21.62), bacillolysin (EC 3.4.24.28) serine protease alkaline (EC 3.4.24.28) (EC 3.4.21.x), askeratinases (EC 3.4.x.x), defibration enzymes, cutinases, esterases and β-mannanase (EC 3.2.1.78). 13. Method according to claim 9, characterized in that the polypeptide is used in combination with one or more of the enzymes selected from the group consisting of endoglucanases (EC 3.2.1.4), celiobiohydrolases (EC 3.2.1.91), β-glucosidases (EC 3.2.1.21), cellulases (EC 3.2.1.74), lichenases (EC 3.2.1.73), lipases (EC 3.1.1.3), lipid acyltransferases (EC 2.3.1.x), phospholipases (EC 3.1. 1.4, EC 3.1.1.32 and EC 3.1.1.5), 6-phytase (EC 3.1.3.26), 3-phytase (EC 3.1.3.8), amylases, alpha-amylases (EC 3.2.1.1), xylanases (EC 3.2. 1.8, EC 3.2.1.32, EC 3.2.1.37, EC 3.2.1.72 and EC 3.2.1.136), glucoamylases (EC 3.2.1.3), hemicellulases, subtilisin (EC 3.4.21.62), bacillolysin (EC 3.4.24.28) serine protease alkaline (EC 3.4.24.28) (EC 3.4.21.x), askeratinases (EC 3.4.x.x), defibration enzymes, cutinases, esterases and β-mannanase (EC 3.2.1.78). 14. Polypeptide, according to claim 1, characterized in that it has a residual activity of at least 70% when incubated with 0.2 mg/ml of pepsin in a buffered solution at pH 3.5 for two hours at a temperature of 40°C. 15. Polypeptide according to claim 1, characterized in that it solubilizes arabinoxylans without increasing viscosity in a reaction medium.