Enzymes

Abstract

The present invention relates to new enzymes with improved properties and to compositions comprising these enzymes suitable for use in the production of a food, feed, or malt beverage product, such as in a brewing process.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to enzymes with improved properties and to compositions comprising these enzymes suitable for use in the production of a food, beverage (e.g. beer), feed, or biofuel, such as in a brewing process.

BACKGROUND OF THE INVENTION

[0002] The use of enzymes in beer production is well known. Application of enzymes to the mashing step to improve mash filterability and increase extract yield is described in WO 97/42302.

[0003] WO2005118769 and WO2005059084 relates to a mashing and filtration step in a process for the production of beer, and to enzymatic compositions for use in such a process.

[0004] WO1999057325 relates to strains of Penicillium funiculosum, to new enzyme mixtures obtained from it and nucleic sequences thereto.

[0005] However, there is a need for improved enzymes as well as combination of enzymes useful in the productions of food and beverage products, such as in the mashing, cooking and filtration steps in the production of an alcoholic beverage, such as beer or whiskey.

OBJECT OF THE INVENTION

[0006] It is an object of embodiments of the invention to provide enzymes suitable for the production of food and beverage products, such as in the production of an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey. The enzymes provided may have improved properties in relation to the use in brewing. These wide varieties of improved properties comprise e.g. improved temperature optimums, improved ratio in activity on soluble (WE-AX) to insoluble (WU-AX) arabinoxylan substrates, reduced total pressure built up during lautering and/or filtration steps of a brewing process, as well as increased filterability of enzyme treated material.

SUMMARY OF THE INVENTION

[0007] It has been found by the present inventor(s) that one or more enzyme as well as certain combinations of enzymes have improved properties relative to known enzymes and enzyme combinations, particularly in relation to the use in a process of brewing, wherein starch containing material is treated with the one or more enzyme to produce a brewing mash.

[0008] So, in a first aspect the present invention relates to an enzyme exhibiting endo-1,4-.beta.-xylanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:18, or any functional fragment thereof.

[0009] As used herein "functional fragment" refers to a truncated version of an enzyme with essentially the same or at least a significant degree of enzyme activity as the non-truncated reference enzyme.

[0010] In a second aspect, the present invention relates to an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any functional fragment thereof.

[0011] In a third aspect the present invention relates to a DNA construct comprising a DNA sequence encoding an enzyme according to the invention.

[0012] In a further aspect the present invention relates to a recombinant expression vector comprising a DNA construct comprising a DNA sequence encoding an enzyme according to the invention.

[0013] In a further aspect the present invention relates to a cell that has been transformed with a DNA construct comprising a DNA sequence encoding an enzyme according to the invention.

[0014] In a further aspect the present invention relates to preparation comprising an enzyme, or a DNA construct, or a vector, or a cell according to the invention.

[0015] In a further aspect the present invention relates to composition comprising an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the invention in combination with any one or more .beta.-glucanase.

[0016] In a further aspect the present invention relates to a composition comprising an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to the invention in combination with any one or more xylanase.

[0017] In a further aspect the present invention relates to the use of an enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention in the production of a food, feed, or malt beverage product, such as beer or whiskey.

[0018] In a further aspect the present invention relates to the use of an enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention, in the production of dough or baked products.

[0019] In a further aspect the present invention relates to the use of an enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention, in the preparation of pulp or paper.

[0020] In a further aspect the present invention relates to the use of an enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention, for the preparation of cereal components. In some embodiments the cereal is rye, wheat, or barley.

[0021] In a further aspect the present invention relates to the use of enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention, in the production of beer or modification of by-products from a brewing process.

[0022] In a further aspect the present invention relates to the use of enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention, in the production of wine or juice.

[0023] In a further aspect the present invention relates to the use of enzyme according to the invention, or a preparation according to the invention, or a composition according to the invention, in the production of a first- or second-generation biofuel, such as bioethanol.

[0024] In a further aspect the present invention relates to a method of altering filterability of a starch comprising material, said method comprising the step of treating said starch comprising material with enzyme, or a preparation, or a composition according to the invention.

[0025] In a further aspect the present invention relates to a method of reducing pressure built up during lautering in a brewing application, said method comprising the step of treating a brewing mash with enzyme, or a preparation, or a composition according to the invention.

[0026] In a further aspect the present invention relates to a method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, said method comprising the step of treating a starch comprising material with enzyme, or a preparation, or a composition according to the invention.

[0027] In a further aspect the present invention relates to a method for the production of a brewing mash, said method comprising the step of treating a starch comprising material with an enzyme, or a preparation, or a composition according to the invention.

[0028] In a further aspect the present invention relates to a method for the production of a first- or second-generation biofuel, such as bioethanol, said method comprising the step of treating a starch comprising material with an enzyme, or a preparation, or a composition according to the invention.

[0029] In a further aspect the present invention relates to a product obtained by a method according to the invention.

[0030] In a further aspect the present invention relates to a composition comprising the product obtained by a method according to the invention, such as wherein the product is in a range of 0.1%-99.9%.

LEGENDS TO THE FIGURE

[0031] FIG. 1: Mashing profile used in lab scale and pilot scale brewing. Mashing was initiated by a 10 min mashing-in period after which the enzyme was added.

[0032] FIG. 2: Pilot scale Brewing application results from verification of the glucanase and xylanases screening. The B. sub glucanase S combined with the A. tub xylanases was tested against a blank and UltraFlo max. Data collected was the average flow (L/h), the total pressure build up over the lautering (mm WC, where 1 mm WC=9.80665 Pa) and the max pressure recorded during the lautering (mm WC).

[0033] FIG. 3: Xylanase functionality in brewing

[0034] FIG. 4: Flow--lautering applying various xylanase candidates

[0035] FIG. 5: Beer filtration--average of repeated filtrations

[0036] FIG. 6: Beer filtration--average of repeated filtrations

[0037] FIG. 7: Mashing diagram of example 3.

DETAILED DISCLOSURE OF THE INVENTION

[0038] Beer is traditionally referred to as an alcoholic beverage derived from malt, such as malt derived from barley grain, and optionally adjunct, such as starch containing plant material (e.g. cereal grains) and optionally flavoured, e.g. with hops.

[0039] In the context of the present invention, the term "beer" is meant to comprise any fermented wort, produced by fermentation/brewing of a starch-containing plant material, thus in particular also beer produced exclusively from adjunct, or any combination of malt and adjunct.

[0040] The term "fermentation" means in the present context production of a substance such as ethanol by growing microorganisms in a culture. Commonly, microorganisms such as yeast are used for fermentation.

[0041] As used herein the term "malt" is understood as any malted cereal grain, such as malted barley. "Adjunct" can be defined as any starch-containing plant material which is not malt or barley malt.

[0042] "Starch-containing plant material" can e.g. be one or more cereal, such as barley, wheat, maize, rye, sorghum, millet, or rice, and any combination thereof. The starch-containing plant material can be processed, e.g. milled, malted, partially malted or unmalted. Unmalted cereal is also called "raw grain". Examples of non-cereal starch-containing plant material comprise e.g. tubers, such as potatoes and cassava.

[0043] As used herein, the terms "beverage" and "beverage(s) product" includes such foam forming fermented beverages as full malted beer, beer brewed under the "Reinheitsgebot", ale, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor and the like. The term "beverages" or "beverages product" also includes non-foaming beer and alternative malt beverages such as fruit flavoured malt beverages, e. g., citrus flavoured, such as lemon-, orange-, lime-, or berry-flavoured malt beverages, liquor flavoured malt beverages, e. g., vodka-, rum-, or tequila-flavoured malt liquor, or coffee flavoured malt beverages, such as caffeine-flavoured malt liquor, and the like.

[0044] Beer can be made from a variety of starch-containing plant material by essentially the same process, where the starch is consists mainly of glucose homopolymers in which the glucose residues are linked by either alpha-1, 4- or alpha-1,6-bonds, with the former predominating.

[0045] The process of making fermented beverages such as beer is commonly referred to as brewing. The traditional raw materials used in making these beverages are water, hops and malt. In addition or instead of malt, adjuncts such as common corn grits, refined corn grits, brewer's milled yeast, rice, sorghum, refined corn starch, barley, barley starch, dehusked barley, wheat, wheat starch, torrified cereal, cereal flakes, rye, oats, potato, tapioca, and syrups, such as corn syrup, sugar cane syrup, inverted sugar syrup, barley and/or wheat syrups, and the like may be used as a source of starch. The starch will eventually be converted enzymatically into fermentable sugars.

[0046] Concerning beers made predominantly from malt (e.g. up to 15-20% adjunct), for a number of reasons, the malt, which is produced principally from selected varieties of barley, has the greatest effect on the overall character and quality of the beer. First, the malt is the primary flavouring agent in beer. Second, the malt provides the major portion of the fermentable sugar. Third, the malt provides the proteins, which will contribute to the body and foam character of the beer. Fourth, the malt provides the necessary enzymatic activity during mashing.

[0047] Hops also contribute significantly to beer quality, including flavouring. In particular, hops (or hops constituents) add desirable bittering substances to the beer. In addition, the hops act as protein precipitants, establish preservative agents and aid in foam formation and stabilization. Not all beers are produced using hops. Other stabilizing agents, such as proteases (e.g. papain) may also be used.

[0048] Without Wanting to be Construed as Limiting for the Present Invention, a Conventional Brewing Process can be Described as Follows:

[0049] The process for making beer is well known in the art, but briefly, it involves five steps: (a) mashing and/or adjunct cooking (b) wort separation and extraction (c) boiling and hopping of wort (d) cooling, fermentation and storage, and (e) maturation, processing and packaging. Typically, in the first step, milled or crushed malt is mixed with water and held for a period of time under controlled temperatures to permit the enzymes present in the malt to convert the starch present in the malt into fermentable sugars.

[0050] In the second step, the mash is transferred to a "lauter tun" or mash filter where the liquid is separated from the grain residue. This sweet liquid is called "wort" and the left over grain residue is called "spent grain". The mash is typically subjected to an extraction, which involves adding water to the mash in order to recover the residual soluble extract from the spent grain.

[0051] In the third step, the wort is boiled vigorously. This sterilizes the wort and helps to develop the colour, flavour and odour. Hops are added at some point during the boiling.

[0052] In the fourth step, the wort is cooled and transferred to a fermentor, which either contains the yeast or to which yeast is added. The yeast converts the sugars by fermentation into alcohol and carbon dioxide gas; at the end of fermentation the fermentor is chilled or the fermentor may be chilled to stop fermentation. The yeast flocculates and is removed.

[0053] In the last step, the beer is cooled and stored for a period of time, during which the beer clarifies and its flavour develops, and any material that might impair the appearance, flavour and shelf life of the beer settles out. Prior to packaging, the beer is carbonated and, optionally, filtered and pasteurized.

[0054] After fermentation, a beverage is obtained which usually contains from about 2% to about 10% alcohol by weight. The non-fermentable carbohydrates are not converted during fermentation and form the majority of the dissolved solids in the final beer.

[0055] This residue remains because of the inability of malt amylases to hydrolyze the alpha-1,6-linkages of the starch. The non-fermentable carbohydrates contribute about 50 calories per 12 ounces of beer.

[0056] Recently, there has been a widespread popularization of brewed beverages called light beers, reduced calorie beers or low calorie beers, particularly in the U. S. market. As defined in the U. S., these beers have approximately 30% fewer calories than a manufacturer's "normal" beer.

[0057] Further information on conventional brewing processes, as well as definitions for terms used in the field of brewing technology to be applied for the present invention, may be found in "Technology Brewing and Malting" by Wolfgang Kunze of the Research and Teaching Institute of Brewing, Berlin (VLB), 2nd revised Edition 1999, ISBN 3-921690-39-0, 3rd edition (2004): ISBN 3-921690-49-8, 4.sup.th updated edition, 2010 (ISBN 978-3-921690-64-2).

[0058] Xylanases are classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156; their activity may be measured e.g. as described in the examples. Suitable xylanases to be used in combination with an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to the invention includes any xylanase classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156, such as any one disclosed in WO 2010072226, WO 2010072225, WO 2010072224, WO 2005059084, WO2007056321, WO2008023060A, WO9421785, WO2006114095, WO2006066582, US 2008233175, and WO10059424.

[0059] Endo-1,4-beta xylanase is classified as EC 3.2.1.8. The enzyme causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.

[0060] The terms "family 11 xylanase", "Glycoside hydrolase (GH) family 11" or simply "GH 11 xylanase" as used herein refers to an endo-1,4-beta xylanase classified as EC 3.2.1.8, which causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans and which is classified as a family 11 xylanase according to B. Henrissat, A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280 (1991), pp. 309-316.

[0061] The terms "Family 10 xylanase", "Glycoside hydrolase (GH) family 10", or simply "GH 10 xylanase" comprises enzymes with a number of known activities, such as xylanase (EC:3.2.1.8); endo-1,3-beta-xylanase (EC:3.2.1.32); cellobiohydrolase (EC:3.2.1.91). These enzymes were formerly known as cellulase family F.

[0062] In some embodiments the enzyme exhibiting endo-1,4-.beta.-xylanase activity is a family 11 xylanase. In some embodiments the enzyme exhibiting endo-1,4-.beta.-xylanase activity is a family 10 xylanase.

[0063] In one aspect, the enzyme composition according to the invention has endo-1,4-beta xylanase activity as measured by the assay described in the examples.

[0064] An assay for measuring xylanase activity may be carried out at pH 3.5 or pH 5 and 50.degree. C. using xylan as substrate, or it can be performed at different pH and temperature values for the additional characterisation and specification of enzymes. Enzyme activity is calculated from the increase in absorbance caused by xylose at 540 nm per unit time.

[0065] In some embodiments the enzyme composition according to the invention comprises a xylanase activity of at least about 5000 U/g, such as at least about 6000 U/g, such as at least about 7000 U/g, such as at least about 8000 U/g, such as at least about 8500 U/g, as measured by in the assay described in the examples.

[0066] The enzyme composition according to the invention may have cellulolytic activity. The systematic name of cellulose is 4-(1,3;1,4)-.beta.-D-glucan 4-glucanohydrolase and cellulolytic enzymes or cellulases are classified in EC 3.2.1.4. Cellulase endohydrolyse (1.fwdarw.4)-.beta.-D-glucosidic linkages in e.g. cellulose, lichenin and cereal .beta.-D-glucans and will also hydrolyse 1,4-linkages in .beta.-D-glucans also containing 1,3-linkages. Cellulase also have other names such as endo-1,4-.beta.-D-glucanase, .beta.-1,4-glucanase, .beta.-1,4-endoglucan hydrolase, cellulase A, cellulosin AP, endoglucanase D, alkali cellulose, cellulase A 3, celludextrinase, 9.5 cellulase, avicelase, pancellase SS and 1,4-(1,3;1,4)-.beta.-D-glucan 4-glucanohydrolase.

[0067] In one aspect of the invention, the cellulase activity of the enzyme composition according to the invention is measured by the "Cellulase activity method" as described in the following under the heading "Assays".

[0068] In further aspects, the present invention relates to enzymes having endo-1,3(4)-.beta.-glucanase activity is determined by the assay described in the examples.

[0069] ".beta.-glucanase" or "beta-glucanase" as used herein refers to an endo-1,3(4)-beta-glucanase of EC 3.2.1.6. Catalyze the endohydrolysis of (1->3)- or (1->4)-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3. Suitable beta-glucanases to be used in combination with an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the invention includes any one beta-glucanase disclosed in WO2004087889, WO2005059084, WO9414953, WO2007056321, WO9531533, WO08023060, WO2005100582, WO9828410, WO9742301, WO2006066582, WO05118769, WO2005003319, and WO10059424.

[0070] The standard assay is carried out at pH 5.0, and it can be performed at different pH values for the additional characterisation and specification of enzymes.

[0071] One unit of endo-1,3(4)-.beta.-glucanase activity is defined as the amount of enzyme which produces 1 .mu.mole glucose equivalents per minute under the conditions of the assay (pH 5.0 (or as specified) and 50.degree. C.).

[0072] In some embodiments the enzyme composition according to the invention comprises a .beta.-glucanase activity of at least about 10000 U/g, such as at least about 12000 U/g, such as at least about 14000 U/g, such as at least about 15000 U/g, such as at least about 18000 U/g as measured by the assay described in the examples.

[0073] In further aspects, the enzyme composition according to the invention has laminarinase activity or comprises any one or more further enzyme having laminarinase activity. The laminarinase activity is determined as described in the laminarase assay described in the Assay section.

[0074] Laminarinase may be an endo-1,3(4)-beta-glucanase classified in E.C. 3.2.1.6 or glucan endo-1,3-beta-D-glucosidase classified in E.C. 3.2.1.39. Endo-1,3(4)-beta-glucanase with the alternative names, laminarinase, endo-1,3-beta-glucanase, Endo-1,4-beta-glucanase is classified in E.C. 3.2.1.6. The substrates include laminarin, lichenin and cereal D-glucans and the enzyme catalyse endohydrolysis of (1->3)- or (1->4)-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3. Glucan endo-1,3-beta-D-glucosidase with the alternative names (1->3)-beta-glucan endohydrolase, Endo-1,3-beta-glucanase and laminarinase is classified in E.C. 3.2.1.39 and hydrolyse (1->3)-beta-D-glucosidic linkages in (1->3)-beta-D-glucans in substrates as eg. laminarin, paramylon and pachyman.

[0075] In some aspects, the enzyme composition according to the invention has arabinanase activity or comprises a further enzyme having arabinanase activity. Arabinanase is classified as EC 3.2.1.99. The systematic name is 5-.alpha.-L-arabinan 5-.alpha.-L-arabinanohydrolase but it has several other names such as arabinan endo-1,5-.alpha.-L-arabinosidase, and endo-1,5-.alpha.-L-arabinanase, endo-.alpha.-1,5-arabanase, endo-arabanase, 1,5-.alpha.-L-arabinan and 1,5-.alpha.-L-arabinanohydrolase. Arabinase endohydrolyses (1.fwdarw.5)-.alpha.-arabinofuranosidic linkages in (1.fwdarw.5)-arabinans. Arabinanase also acts on arabinan.

[0076] In one aspect of the invention, the arabinase activity of the enzyme composition according to the invention is measured by arabinase assay as described in the following under the heading "Assays". The assay can be carried out at pH 3.5 and 50.degree. C. using sugar beet arabinan as substrate, and it can be performed at different pH and temperature values for the additional characterisation and specification of enzymes. Enzyme activity is calculated from the increase in absorbance at 540 nm per unit time.

[0077] One unit of arabinase activity is defined as the amount of enzyme (normalised for total assay volume) that gives an increase in .DELTA.OD.sub.540 nmmin.sup.-1 under the conditions of the assay (pH 3.5 and 50.degree. C.).

[0078] In some aspects, the enzyme composition according to the invention has beta-D-glucoside glucohydrolase activity or comprises a further enzyme having beta-D-glucoside glucohydrolase activity. Beta-D-glucoside glucohydrolase refers to enzymes of E.C 3.2.1.21.

[0079] In some aspects, the enzyme composition according to the invention has .beta.-Xylosidase activity or comprises a further enzyme having .beta.-Xylosidase activity. ".beta.-Xylosidase" or "Xylan 1,4-beta-xylosidase" refers to enzymes of E.C 3.2.1.37. .beta.-Xylosidase catalyze the hydrolysis of (1->4)-beta-D-xylans, to remove successive D-xylose residues from the non-reducing termini.

[0080] In some aspects of the invention, the enzyme composition according to the invention has cellobiohydrolase activity or comprises a further enzyme having cellobiohydrolase activity. "Cellobiohydrolase" or "Cellulose 1,4-beta-cellobiosidase" refers to enzymes of EC 3.2.1.91. Cellulose 1,4-beta-cellobiosidase catalyze hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains.

[0081] The cellobiohydrolase activity of the enzyme composition according to the invention is measured by the cellobiohydrolase assay as described in the following under the heading "Assays". The standard assay is carried out at pH 5.0, and it can be performed at different pH values for the additional characterisation and specification of enzymes.

[0082] One unit of cellobiohydrolase activity is defined as the amount of enzyme which produces 1 .mu.mole p-nitrophenol from p-nitrophenyl .beta.-D-cellobiopyranoside per minute under the conditions of the assay (pH 5.0 (or as specified) and 50.degree. C.).

[0083] In some aspects, the enzyme composition according to the invention has .alpha.-N-arabinofuranosidase activity or comprises a further enzyme having arabinofuranosidase activity. ".alpha.-N-arabinofuranosidase" or "Alpha-N-arabinofuranosidase" refers to enzymes of EC 3.2.1.55. .alpha.-N-arabinofuranosidase catalyze the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides.

[0084] In one aspect of the invention, the arabinofuranosidase activity of the enzyme composition according to the invention is measured by the arabinofuranosidase assay as described in the following under the heading "Assays". The standard assay can be carried out at pH 5.0 and 50.degree. C. and it can be performed at different values of pH and temperature for the additional characterisation and specification of enzymes.

[0085] One unit of .alpha.-N-arabinofuranosidase activity is defined as the amount of enzyme which produces 1 .mu.mole p-nitrophenol from p-nitrophenyl .alpha.-L-arabinofuranoside per minute under the conditions of the assay (pH 5.0 and 50.degree. C. (or as specified)).

[0086] In some aspects, the enzyme composition according to the invention has glucan 1,4-beta-glucosidase activity or comprises a further enzyme having glucan 1,4-beta-glucosidase activity. "Glucan 1,4-beta-glucosidase" or "glucan 1,4-beta-glucosidase" refers to enzymes of E.C3.2.1.74. Glucan 1,4-beta-glucosidase catalyze the hydrolysis of (1->4)-linkages in (1->4)-beta-D-glucans, to remove successive glucose units.

[0087] In some aspects, the enzyme composition according to the invention has xyloglucan-specific exo-beta-1,4-glucanase activity or comprises a further enzyme having xyloglucan-specific exo-beta-1,4-glucanase activity. "xyloglucan-specific exo-beta-1,4-glucanase" refers to enzymes of E.C3.2.1.155. Xyloglucan-specific exo-beta-1,4-glucanase catalyze the exohydrolysis of (1->4)-beta-D-glucosidic linkages in xyloglucan.

[0088] The enzymes and enzyme compositions according to the proceeding aspects may be used in a process comprising reducing the viscosity of an aqueous solution comprising a starch hydrolysate.

[0089] The enzymes and enzyme compositions may also be used in a process comprising filtering of an aqueous solution comprising a starch hydrolysate. In some embodiments the aqueous solution comprising a starch hydrolysate is a mash for beer making, and in other embodiments the aqueous solution comprising a starch hydrolysate is a food composition.

[0090] Alternatively, the enzyme composition according to the present invention may be used in the production of fruit juice, wine, grain processing, fuel alcohol, first- or second-generation biofuel, such as bioethanol, and potable alcohol.

[0091] In some embodiments the first- or second-generation biofuel, such as bioethanol is produced from agricultural feed stocks such as sugar cane, potato, corn, wheat sorghum etc. or from cellulosic material such as corn stover, switchgrass or other plant material. In both cases fermentable sugars are extracted from the raw material and fermented by microorganisms into alcohol, which is distilled and may be used as transportation fuel. The enzyme composition according to the present invention may be used in this production of biofuel. The enzymes complex may be added to enhance extraction of polysaccharides from the raw material, help degrade polysaccharides down into fermentable sugars and/or to enhance processing parameters such as separation of liquids from solids, flow characteristics and pumpability.

[0092] The process of the invention may be applied in the mashing of any grist. According to the invention the grist may comprise any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers, roots, stems, leaves and seeds.

[0093] In some embodiments the grist comprises grain, such as grain from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from grain.

[0094] In some embodiments the grist comprises malted grain, such as barley malt. Preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from malted grain.

[0095] The term "mash" is understood as aqueous starch slurry, e. g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, mixed with water later to be separated into wort+spent grains.

[0096] The term "mash separation" is understood as the separation of wort from spent grains, such as by lautering or mash filtration.

[0097] The term "beer filtration" is understood as a separation process in which the yeast cells and other turbidity-causing materials still present in the beer are removed, such as by microfiltration or membrane processes.

[0098] The enzyme preparation, such as in the form of a food ingredient prepared according to the present invention, may be in the form of a solution or as a solid--depending on the use and/or the mode of application and/or the mode of administration. The solid form can be either as a dried enzyme powder or as a granulated enzyme.

[0099] In one aspect the invention provides an enzyme composition preparation comprising the enzyme or enzyme composition according to the invention, an enzyme carrier and optionally a stabilizer and/or a preservative.

[0100] In yet a further aspect of the invention, the enzyme carrier is selected from the group consisting of glycerol or water.

[0101] In a further aspect, the preparation comprises a stabilizer. In one aspect, the stabilizer is selected from the group consisting of inorganic salts, polyols, sugars and combinations thereof. In one aspect, the stabilizer is an inorganic salt such as potassium chloride. In another aspect, the polyol is glycerol, propylene glycol, or sorbitol. In yet another aspect, the sugar is a small-molecule carbohydrate, in particular any of several sweet-tasting ones such as glucose, fructose and saccharose.

[0102] In yet at further aspect, the preparation comprises a preservative. In one aspect, the preservative is methyl paraben, propyl paraben, benzoate, sorbate or other food approved preservatives or a mixture thereof.

Specific Embodiments of the Invention

[0103] In some embodiments the enzyme exhibiting endo-1,4-.beta.-xylanase activity, optionally in combination with any one or more .beta.-glucanase according to the present invention provides for a significantly reduced viscosity in brewing applications facilitating improved mash and beer separation.

[0104] Desired xylanase characteristics for brewing applications may include one or more of the following aspects: [0105] a) Enzyme substrate specificity [0106] WE-AX/WU-AX ratio has an impact on viscosity. In some embodiments this ratio is less than about 7.0, such as less than about 6.5, such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about 4.5. [0107] b) Enzyme substrate selectivity [0108] How close to branch points the enzyme(s) cuts is believed to have an impact on the functionality. [0109] c) Enzyme thermostability [0110] Continuous solubilisation of AX during mashing--thermostability a key feature. Accordingly, in some embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the present invention is thermostable within a temperature range of 65-78.degree. C. [0111] d) Enzyme pH optimum. Accordingly, in some embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase activity has a pH optimum in the range of pH 5.4-5.6. [0112] e) Enzyme inhibition (e.g. known key factor for xylanases)

[0113] Said significantly reduced viscosity in brewing applications may be measured as a reduced viscosity in the brewing application as compared to a control with a known enzyme or combination of enzyme activities, such as Ultraflo.RTM. Max used under same conditions and amounts.

[0114] In some embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the present invention, optionally in combination with any one or more .beta.-glucanase according to the present invention provides for an improved mash and beer separation in brewing applications.

[0115] In some embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the present invention, optionally in combination with any one or more .beta.-glucanase according to the present invention provides for a low potential for off flavour formation, such as off flavour formation related to arabinoxylan breakdown.

[0116] In some embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the present invention, optionally in combination with any one or more .beta.-glucanase according to the present invention provides for a decreased risk of filter bed collapse, such as at lautering.

[0117] In some embodiments, the enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the present invention, optionally in combination with any one or more .beta.-glucanase according to the present invention provides for a reduction in off flavour potential and/or reduction in off flavor formation. One aspect of the invention relates to an enzyme exhibiting endo-1,4-.beta.-xylanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:18, or any functional fragment thereof.

[0118] Another aspect relates to an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any functional fragment thereof.

[0119] In some embodiments of the invention the enzyme exhibiting endo-1,4-.beta.-xylanase activity has a ratio in activity on soluble arabinoxylan substrate (WE-AX) to insoluble arabinoxylan substrate (WU-AX) arabinoxylan substrate of less than about 7.0, such as less than about 6.5, such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about 4.5.

[0120] In some embodiments the enzyme according to the invention has a temperature optimum in the range of 40-70.degree. C., such as in the range of 45-65.degree. C., such as in the range of 50-65.degree. C., such as in the range of 55-65.degree. C.

[0121] In some embodiments the enzyme according to the invention has at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

[0122] In some embodiments the enzyme according to the invention has a total number of amino acids of less than 350, such as less than 340, such as less than 330, such as less than 320, such as less than 310, such as less than 300 amino acids, such as in the range of 200 to 350, such as in the range of 220 to 345 amino acids.

[0123] In some embodiments the amino acid sequence of said enzyme according to the invention has at least one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions as compared to any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

[0124] In some embodiments the amino acid sequence of said enzyme according to the invention has a maximum of one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions compared to any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

[0125] In some embodiments the enzyme according to the invention comprises the amino acid sequence identified by any one of SEQ ID NO: 1-18, or any functional fragment thereof.

[0126] In some embodiments the enzyme according to the invention consists of the amino acid sequence identified by any one of SEQ ID NO: 1-18, or any functional fragment thereof.

[0127] A further important aspect of the invention relates to a composition comprising an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the invention in combination with any one or more .beta.-glucanase. In some embodiments this one or more .beta.-glucanase is according to the invention.

[0128] A further important aspect of the invention is a composition comprising an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to the invention in combination with any one or more xylanase. In some embodiments this one or more xylanase is an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to the invention. In some embodiments this one or more xylanase is an enzyme according to SEQ ID NO:17 and/or SEQ ID NO:18, or any functional fragment thereof.

[0129] In some embodiments the combination of an enzyme exhibiting endo-1,4-.beta.-xylanase activity with an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity is according to the following table:

TABLE-US-00001 1.sup.st enzyme (Xylanase) 2.sup.nd enzyme SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (Glucanase) NO: 1 NO: 2 NO: 3 NO: 4 NO: 5 NO: 6 NO: 17 NO: 18 SEQ ID NO: 7 X X X X X X X X SEQ ID NO: 8 X X X X X X X X SEQ ID NO: 9 X X X X X X X X SEQ ID NO: 10 X X X X X X X X SEQ ID NO: 11 X X X X X X X X SEQ ID NO: 12 X X X X X X X X SEQ ID NO: 13 X X X X X X X X SEQ ID NO: 14 X X X X X X X X SEQ ID NO: 15 X X X X X X X X SEQ ID NO: 16 X X X X X X X X

[0130] It is to be understood that any one of the above combination of a 1.sup.st enzyme being an enzyme exhibiting endo-1,4-.beta.-xylanase activity may be combined with one one enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity with a ratio between the two enzymes of 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 10:10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, or 10:1, such as within a range of 1:10-10:1, such as 2:10-10:2, such as 3:10-10:3, such as 4:10-10:4, such as 5:10-10:5, such as 6:10-10:6, such as 7:10-10:7, such as 8:10-10:8, or within 9:10-10:9.

[0131] In some embodiments the composition according to the invention comprises a combination of at least two enzymes, said two enzymes, or two enzymes with an amino acid sequence having at least 80% sequence identity with the respective SEQ ID, or any functional fragment thereof, being selected from the list consisting of

SEQ ID NO:1 and SEQ ID NO:7;

SEQ ID NO:2 and SEQ ID NO:7;

SEQ ID NO:3 and SEQ ID NO:7;

SEQ ID NO:4 and SEQ ID NO:7;

SEQ ID NO:5 and SEQ ID NO:7;

SEQ ID NO:6 and SEQ ID NO:7;

SEQ ID NO:17 and SEQ ID NO:7;

SEQ ID NO:18 and SEQ ID NO:7;

SEQ ID NO:1 and SEQ ID NO:8;

SEQ ID NO:2 and SEQ ID NO:8;

SEQ ID NO:3 and SEQ ID NO:8;

SEQ ID NO:4 and SEQ ID NO:8;

SEQ ID NO:5 and SEQ ID NO:8;

SEQ ID NO:6 and SEQ ID NO:8;

SEQ ID NO:17 and SEQ ID NO:8;

SEQ ID NO:18 and SEQ ID NO:8;

SEQ ID NO:1 and SEQ ID NO:9;

SEQ ID NO:2 and SEQ ID NO:9;

SEQ ID NO:3 and SEQ ID NO:9;

SEQ ID NO:4 and SEQ ID NO:9;

SEQ ID NO:5 and SEQ ID NO:9;

SEQ ID NO:6 and SEQ ID NO:9;

SEQ ID NO:17 and SEQ ID NO:9;

SEQ ID NO:18 and SEQ ID NO:9;

SEQ ID NO:1 and SEQ ID NO:10;

SEQ ID NO:2 and SEQ ID NO:10;

SEQ ID NO:3 and SEQ ID NO:10;

SEQ ID NO:4 and SEQ ID NO:10;

SEQ ID NO:5 and SEQ ID NO:10;

SEQ ID NO:6 and SEQ ID NO:10;

SEQ ID NO:17 and SEQ ID NO:10;

SEQ ID NO:18 and SEQ ID NO:10;

SEQ ID NO:1 and SEQ ID NO:11;

SEQ ID NO:2 and SEQ ID NO:11;

SEQ ID NO:3 and SEQ ID NO:11;

SEQ ID NO:4 and SEQ ID NO:11;

SEQ ID NO:5 and SEQ ID NO:11;

SEQ ID NO:6 and SEQ ID NO:11;

SEQ ID NO:17 and SEQ ID NO:11;

SEQ ID NO:18 and SEQ ID NO:11;

SEQ ID NO:1 and SEQ ID NO:12;

SEQ ID NO:2 and SEQ ID NO:12;

SEQ ID NO:3 and SEQ ID NO:12;

SEQ ID NO:4 and SEQ ID NO:12;

SEQ ID NO:5 and SEQ ID NO:12;

SEQ ID NO:6 and SEQ ID NO:12;

SEQ ID NO:17 and SEQ ID NO:12;

SEQ ID NO:18 and SEQ ID NO:12;

SEQ ID NO:1 and SEQ ID NO:13;

SEQ ID NO:2 and SEQ ID NO:13;

SEQ ID NO:3 and SEQ ID NO:13;

SEQ ID NO:4 and SEQ ID NO:13;

SEQ ID NO:5 and SEQ ID NO:13;

SEQ ID NO:6 and SEQ ID NO:13;

SEQ ID NO:17 and SEQ ID NO:13;

SEQ ID NO:18 and SEQ ID NO:13;

SEQ ID NO:1 and SEQ ID NO:14;

SEQ ID NO:2 and SEQ ID NO:14;

SEQ ID NO:3 and SEQ ID NO:14;

SEQ ID NO:4 and SEQ ID NO:14;

SEQ ID NO:5 and SEQ ID NO:14;

SEQ ID NO:6 and SEQ ID NO:14;

SEQ ID NO:17 and SEQ ID NO:14;

SEQ ID NO:18 and SEQ ID NO:14;

SEQ ID NO:1 and SEQ ID NO:15;

SEQ ID NO:2 and SEQ ID NO:15;

SEQ ID NO:3 and SEQ ID NO:15;

SEQ ID NO:4 and SEQ ID NO:15;

SEQ ID NO:5 and SEQ ID NO:15;

SEQ ID NO:6 and SEQ ID NO:15;

SEQ ID NO:17 and SEQ ID NO:15;

SEQ ID NO:18 and SEQ ID NO:15;

SEQ ID NO:1 and SEQ ID NO:16;

SEQ ID NO:2 and SEQ ID NO:16;

SEQ ID NO:3 and SEQ ID NO:16;

SEQ ID NO:4 and SEQ ID NO:16;

SEQ ID NO:5 and SEQ ID NO:16;

SEQ ID NO:6 and SEQ ID NO:16;

SEQ ID NO:17 and SEQ ID NO:16; and

SEQ ID NO:18 and SEQ ID NO:16.

[0132] In some embodiments the endo-1,3(4)-.beta.-glucanase activity and the endo-1,4-.beta.-xylanase activity are derived from at least two different enzymes, such as at least two different enzymes from two different species.

[0133] In some embodiments the total pressure built up is reduced to a value of less than 470 mm WC, such as less than 450 mm WC, such as less than 430 mm WC, such as less than 410 mm WC, such as less than 390 mm WC, such as less than 370 mm WC, such as less than 350 mm WC, such as less than 330 mm WC, such as less than 310 mm WC, such as less than 300 mm WC, such as less than 290 mm WC, when the composition according to the present invention is used prior to the lautering in a brewing application.

[0134] In some embodiments the total pressure built up is reduced by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95% compared to the use of a negative control without said composition; when used prior to the lautering in a brewing application.

[0135] In some embodiments the wort filterability as measured by volume wort collected after 5 min of filtration relative to a control without enzymes is increased to above 1.5, such as above 1.6, such as above 1.7, such as above 1.8, such as above 1.9, such as above 2.0, such as above 2.1, such as above 2.2, such as above 2.3, such as above 2.4, such as above 2.5, when the composition according to invention is used in a brewing application prior to the wort separation.

[0136] In some embodiments the wort filterability as measured by volume wort collected after 5 min of filtration is increased at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300% as compared to the use of a negative control without said composition.

[0137] In some embodiments the composition according to the invention comprises any one or more further enzyme. In some embodiments the one or more further enzyme is selected from list consisting of a xylanase classified in EC 3.2.1.32, EC 3.2.1.136, or EC 3.2.1.156, a cellulase, a laminarinase, an endo-1,5-.alpha.-L-arabinanase, a beta-D-glucoside glucohydrolase, a .beta.-Xylosidase, a cellobiohydrolase, a glucan 1,4-beta-glucosidase, a xyloglucan-specific exo-beta-1,4-glucanase and an .alpha.-N-Arabinofuranosidase.

[0138] Sequences and enzymes identified by a sequence as mentioned herein and used according to the present invention alone or in combinations with other enzymes or compounds may be with or without signal peptide.

[0139] Assays

[0140] DNS Cellulase Activity Method (DNS CMC Method)

[0141] Systematic Name: 1,4-(1,3;1,4)-.beta.-D-glucan 4-glucanohydrolase

[0142] IUB Number: EC 3.2.1.4

[0143] Principle

[0144] The assay of cellulase is based on the enzymatic endo-hydrolysis of the 1,4-.beta.-D-glucosidic bonds in carboxymethylcellulose (CMC), a .beta.-1,4-glucan. The products of the reaction (.beta.-1,4 glucan oligosaccharides) was determined colorimetrically by measuring the resulting increase in reducing groups using a 3,5-dinitrosalicylic acid reagent. Enzyme activity was calculated from the relationship between the concentration of reducing groups, as glucose equivalents, and absorbance at 540 nm.

[0145] The assay was carried out at pH 5.0, but it can be performed at different pH values for the additional characterisation and specification of enzymes.

[0146] Unit Definition

[0147] One unit of cellulase activity is defined as the amount of enzyme which produces 1 .mu.mole glucose equivalents per minute under the conditions of the assay (pH 5.0 (or as specified) and 50.degree. C.).

[0148] Materials

[0149] Carboxymethylcellulose. Supplier: Megazyme Ltd. Product no.: CM-Cellulose 4M

[0150] D-Glucose `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10117. M.W.: 180.16

[0151] Sodium acetate anhydrous `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10236. M.W.: 82.03

[0152] Acetic acid ("glacial") `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10001. M.W.: 60.05

[0153] 3,5-Dinitrosalicylic acid GPR (3,5-dinitro-2-hydroxybenzoic acid). Supplier: Merck Ltd (BDH). Product no.: 28235

[0154] Sodium hydroxide pellets `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10252. M.W.: 40.00

[0155] Potassium sodium (+)-tartrate `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10219. M.W.: 282.22

[0156] 1.5% (w/v solution) Carboxymethylcellulose (CMC) solution in 0.1M sodium acetate buffer, pH 5.0 (substrate solution).

[0157] 3,5-Dinitrosalicylic acid (DNS) solution. 20 g/L of DNS in buffer containing 32 g/L sodium hydroxide pellets, and 600 g/L potassium sodium (+)-tartrate.

[0158] Glucose standard solution (0.50 mg/ml)

[0159] Procedure

[0160] The enzyme composition was diluted into samples and a glucose standard curve as shown in FIG. 2 was made using glucose concentrations of 0, 0.125, 0.25, 0.375, and 0.5 mg/ml.

[0161] 0.25 ml of enzyme solution was mixed with 1.75 ml of the substrate solution (1.5% w/v) at 50.degree. C. and the reaction was stopped after 10 min by addition of DNS solution. This is followed by heating to 95.degree. C. for 5 minutes.

[0162] The optical density was measured at 540 nm (OD.sub.540 nm) of the different samples.

[0163] Calculation

[0164] The enzyme activity is determined from the standard curve as shown in FIG. 2.

[0165] The activity is calculated as follows:

Activity ( u ml - 1 or u g - 1 ) = T - c m .times. A .times. 1 180.16 .times. 10 3 1 V .times. 1 t .times. D ##EQU00001##

where:

T=.DELTA.OD.sub.540 nm TEST

[0166] =OD.sub.540 nm TEST-OD.sub.540 nm BLANK m=gradient of the standard curve (approximately 1.0) c=y axis intercept of the standard curve (always negative and approximately -0.02) 180.16.ident.molecular weight of glucose 10.sup.3.ident.to convert to .mu.moles A.ident.assay volume in ml V.ident.enzyme volume in ml t.ident.assay time in minutes D=actual enzyme dilution factor (e.g. for 1.000 g diluted to 1 litre D=1000)

[0167] Laminarinase (DNS Laminarin Method)

[0168] Principle

[0169] The reaction, catalysed by laminarinase, involves the endohydrolysis of 1,3-glucosidic bonds in 1,3-.beta.-D-glucans. Substrates include laminarin, paramylon and pachyman. The products of the reaction (.beta.-1,3-glucan oligosaccharides) are determined colourimetrically by measuring the resulting increase in reducing groups using a 3,5-dintrosalicylic acid reagent. Enzyme activity is calculated from the relationship between the concentration of reducing groups, as glucose equivalents, and absorbance at 540 nm.

[0170] The assay was carried out at pH 5.0 and 50.degree. C., but it can be performed at different values of pH and temperature for the additional characterisation and specification of enzymes.

[0171] Unit Definition

[0172] One unit of laminarinase activity is defined as the amount of enzyme which produces 1 .mu.mole glucose equivalents per minute under the conditions of the assay (pH 5.0 and 50.degree. C. (or as specified)).

[0173] Materials

[0174] See materials given above for the Cellulase activity assay.

[0175] Laminarin (from Laminaria digitata). Supplier: Sigma-Aldrich Co. Ltd. Product no.: L 9634

[0176] 1.00% (w/v solution) Laminarin solution (substrate solution 0.1M sodium acetate buffer, pH 5.0)

[0177] 1.75 ml laminarin solution is mixed with 0.25 ml diluted enzyme solution at 50.degree. C. for 10 minutes and the reaction stopped by addition of 2 ml DNS solution.

[0178] Standard curve was made using 0, 0.125, 0.25, 0.5 and 0.75 mg/ml glucose solution.

[0179] Optical density was measured at 540 nm (OD.sub.540 nm).

[0180] Calculation

[0181] The activity is calculated as follows:

Activity ( u ml - 1 or u g - 1 ) = T - c m .times. A .times. 1 180.16 .times. 10 3 .times. 1 V .times. 1 t .times. D ##EQU00002## [0182] where:

T=.DELTA.OD.sub.540 nm TEST

[0182] [0183] =OD.sub.540 nm TEST-OD.sub.540 nm BLANK m=gradient of the standard curve (approximately 1.0) c=y axis intercept of the standard curve (always negative and approximately -0.03) 180.16.ident.molecular weight of glucose 10.sup.3.ident.to convert to .mu.moles A.ident.assay volume in ml V.ident.enzyme volume in ml t.ident.assay time in minutes D=enzyme dilution factor (e.g. for 1 g diluted to 1 litre D=1000)

[0184] Arabinase Assay.

[0185] Principle

[0186] The assay of Arabinase activity is based on colorimetrically determination by measuring the resulting increase in reducing groups using a 3,5-dinitrosalicylic acid reagent. Enzyme activity was calculated from the relationship between the concentration of reducing groups, as arabinose equivalents, and absorbance at 540 nm.

[0187] The assay was carried out at pH 3.5, but it can be performed at different pH values for the additional characterisation and specification of enzymes.

[0188] Unit Definition

[0189] One unit of arabinase (Arabinanase (endo-1,5-alpha-L-arabinanase)) activity is defined as the amount of enzyme which produces 1 .mu.mole arabinose equivalents per minute under the conditions of the assay (pH 3.5 (or as specified) and 50.degree. C.).

[0190] Materials

[0191] Megazyme Sugar Beet Arabinan

[0192] Arabinose Sigma A3131 M.W.: 150.1

[0193] Sodium acetate anhydrous `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10236. M.W.: 82.03

[0194] Acetic acid ("glacial") `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10001. M.W.: 60.05

[0195] 3,5-Dinitrosalicylic acid GPR (3,5-dinitro-2-hydroxybenzoic acid). Supplier: Merck Ltd (BDH). Product no.: 28235

[0196] Sodium hydroxide pellets `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10252. M.W.: 40.00

[0197] Potassium sodium (+)-tartrate `AnalaR`. Supplier: Merck Ltd (BDH). Product no.: 10219. M.W.: 282.22

[0198] 1.5% (w/v solution) Arabinan solution in 0.1M sodium acetate buffer, pH 3.5 (substrate solution).

[0199] 3,5-Dinitrosalicylic acid (DNS) solution. 20 g/L of DNS in buffer containing 32 g/L sodium hydroxide pellets, and 600 g/L potassium sodium (+)-tartrate.

[0200] Arabinase standard solution (0.50 mg/ml)

[0201] Procedure

[0202] The enzyme composition was diluted into samples and a glucose standard curve was made using arabinase concentrations of 0, 0.125, 0.25, 0.375, and 0.5 mg/ml.

[0203] 0.25 ml of enzyme solution was mixed with 1.75 ml of the substrate solution (1.5% w/v) at 50.degree. C. and the reaction was stopped after 10 min by addition of DNS solution. Followed by heating to 95.degree. C. for 5 minutes.

[0204] The optical density was measured at 540 nm (OD.sub.540 nm) of the different samples.

[0205] Calculation

[0206] The enzyme activity is determined from the standard curve.

[0207] The activity is calculated as follows:

Activity ( u ml - 1 or u g - 1 ) = T - c m .times. A .times. 1 150.13 .times. 10 3 .times. 1 V .times. 1 t .times. D ##EQU00003##

where:

T=.DELTA.OD.sub.540 nm TEST

[0208] =OD.sub.540 nm TEST-OD.sub.540 nm BLANK m=gradient of the standard curve (approximately 1.0) c=y axis intercept of the standard curve (always negative and approximately -0.02) 150.13.ident.molecular weight of arabinase 10.sup.3.ident.to convert to .mu.moles A.ident.assay volume in ml V.ident.enzyme volume in ml t.ident.assay time in minutes D=actual enzyme dilution factor (e.g. for 1.000 g diluted to 1 litre D=1000)

[0209] Arabinofuranosidase Assay.

[0210] The reaction, catalysed by .alpha.-N-arabinofuranosidase, involves the hydrolysis of the terminal bond, at the non-reducing .alpha.-L-arabinofuranoside residue, of .alpha.-L-arabinosides. The enzyme acts on .alpha.-L-arabinofuranosides, .alpha.-L-arabinans containing (1,3)- and/or (1,5)-linkages, arabinoxylans and arabinogalactans.

[0211] The assay of .alpha.-N-arabinofuranosidase is based upon the enzymatic hydrolysis of p-nitrophenyl .alpha.-L-arabinofuranoside. The assay is a "two-point", rather than a "continuous monitoring", method. The calculation of enzyme activity is based on measurements taken only at the beginning and end of the incubation period. A product of the reaction, p-nitrophenol is determined colourimetrically (after pH adjustment). Enzyme activity is calculated from the relationship between the concentration of p-nitrophenol and absorbance at 400 nm.

[0212] Preparation of Diluted Enzyme Solution:

[0213] Prepare all enzyme solutions, from powder or liquid enzyme preparations, with glass distilled water. Minimise assay dilution errors by avoiding large dilution steps involving small volumes or weights. In making enzyme dilutions it is more accurate, even for a liquid sample, to weigh out the initial enzyme sample. If this is done, in the case of liquid samples it is therefore necessary to measure the specific gravity of the liquid at 20.degree. C.

[0214] As the assay is a "two-point", rather than a "continuous monitoring", method it is important to ensure the linearity within the incubation period with different enzyme systems and conditions. Under the standard assay conditions of substrate concentration, pH, temperature and assay time the assay has been demonstrated to be linear in the range .DELTA.OD.sub.540 nm TEST (T)=0.20-1.50. However, for good practice, the assay is operated within a defined range of .DELTA.OD.sub.540 nm TEST (T)=0.400-0.800.

[0215] Procedure

[0216] Each enzyme sample assay involves three analyses: duplicate test (TEST) analyses and a blank (BLANK) analysis. The procedure given describes the analysis of a single enzyme sample.

TABLE-US-00002 TEST BLANK 0.2M Sodium acetate buffer, pH 5.0 1.00 ml 1.00 ml Glass distilled water 1.00 ml 1.00 ml p-Nitrophenyl-.alpha.-L-arabinofuranoside solution 1.00 ml 1.00 ml

[0217] 0.25 ml diluted enzyme solution was added to the solutions at 50.degree. C., the reaction was stopped after 10 minutes by addition of 4 ml of 0.4M glycine solution, pH 10.8 (stop reagent).

[0218] Absorbance was measured at 400 nm at 25.degree. C. against a water blank. [0219] determine OD.sub.400 nm TEST for the duplicate TESTS measured; [0220] determine OD.sub.400 nm BLANK.

[0221] Calculation

.DELTA. OD 400 nm TEST ( T ) = OD 400 nm TEST - OD 400 nm BLANK ##EQU00004## Units ( mol min - 1 ) = T 18300 .times. V 1000 .times. 10 6 .times. 1 t Activity ( u ml - 1 or u g - 1 ) = Units .times. 1 E .times. D ##EQU00004.2##

where: T=OD400 nm TEST-OD400 nm BLANK 18300=Molar extinction coefficient for p-nitrophenol (1 cm path length) V=7.25 (total liquid volume in test in ml) t=10 (minutes) 1 u=1 .mu.molmin-1 E=0.25 (volume of diluted enzyme sample in ml) D=Enzyme dilution factor e.g. for 1 ml diluted to 1 litre D=1000)

[0222] Cellobiohydrolase Assay.

[0223] Principle

[0224] The reaction, catalysed by cellobiohydrolase, involves the hydrolysis of 1,4-.beta.-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains.

[0225] The assay of cellobiohydrolase is based on the enzymatic hydrolysis of p-nitrophenyl .beta.-D-cellobiopyranoside. The product of the reaction, p-nitrophenol is determined colorimetrically (after pH adjustment). Enzyme activity is calculated from the relationship between the concentration of p-nitrophenol and absorbance at 400 nm.

[0226] The assay is operated within the linear defined range of .DELTA.OD.sub.540 nm TEST (T)=0.400-0.800.

[0227] Procedure

[0228] Each enzyme sample assay involves three analyses: duplicate test (TEST) analyses and a blank (BLANK) analysis. The procedure given describes the analysis of a single enzyme sample.

TABLE-US-00003 TEST BLANK 0.2M Sodium acetate buffer, pH 5.0 1.00 ml 1.00 ml Glass distilled water 1.00 ml 1.00 ml p-Nitrophenyl .beta.-D-cellobiopyranoside solution 1.00 ml 1.00 ml

[0229] 0.25 ml diluted enzyme solution was added to the test solution at 50.degree. C., after 30 minutes 4 ml of 0.4M glycine solution, pH 10.8 (stop reagent) was added to each tube. [0230] Absorbance was measured at 20.degree. C. at 400 nm in a 1 cm glass cuvette against a water blank. [0231] determine OD400 nm TEST for the duplicate TESTS measured; [0232] determine OD400 nm BLANK.

[0233] Calculation

.DELTA. OD 400 nm TEST ( T ) = OD 400 nm TEST - OD 400 nm BLANK ##EQU00005## Units ( mol min - 1 ) = T 18300 .times. V 1000 .times. 10 6 .times. 1 t Activity ( u ml - 1 or u g - 1 ) = Units .times. 1 E .times. D ##EQU00005.2##

where: T=OD.sub.400 nm TEST-OD.sub.400 nm BLANK 18300=Molar extinction coefficient for p-nitrophenol (1 cm path length) V=7.25 (total liquid volume in test in ml) 1000=to convert to litres 10.sub.6=to convert to .mu.moles t=30 (minutes) 1 u=1 .mu.molmin.sup.-1 E=0.25 (volume of diluted enzyme sample in ml) D=Enzyme dilution factor e.g. for 1 ml diluted to 1 litre D=1000)

[0234] Numbered embodiments according to the invention:

[0235] 1. An enzyme exhibiting endo-1,4-.beta.-xylanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:18, or any functional fragment thereof.

[0236] 2. The enzyme according to embodiment 1, which enzyme has a ratio in activity on soluble arabinoxylan substrate (WE-AX) to insoluble arabinoxylan substrate (WU-AX) arabinoxylan substrate of less than about 7.0, such as less than about 6.5, such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about 4.5.

[0237] 3. An enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any functional fragment thereof.

[0238] 4. The enzyme according to any one of embodiment 1-3, which enzyme has a temperature optimum in the range of 40-70.degree. C., such as in the range of 45-65.degree. C., such as in the range of 50-65.degree. C., such as in the range of 55-65.degree. C.

[0239] 5. The enzyme according to any one of embodiments 1-4, wherein said enzyme has at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

[0240] 6. The enzyme according to any one of embodiments 1-5 having a total number of amino acids of less than 350, such as less than 340, such as less than 330, such as less than 320, such as less than 310, such as less than 300 amino acids, such as in the range of 200 to 350, such as in the range of 220 to 345 amino acids.

[0241] 7. The enzyme according to any one of embodiments 1-6, wherein the amino acid sequence of said enzyme has at least one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions as compared to any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

[0242] 8. The enzyme according to any one of embodiments 1-7, wherein the amino acid sequence of said enzyme has a maximum of one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions compared to any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

[0243] 9. The enzyme according to any one of embodiments 1-8, which enzyme comprises the amino acid sequence identified by any one of SEQ ID NO: 1-18, or any functional fragment thereof.

[0244] 10. The enzyme according to any one of embodiments 1 or 3, which enzyme consists of the amino acid sequence identified by any one of SEQ ID NO: 1-18, or any functional fragment thereof.

[0245] 11. A DNA construct comprising a DNA sequence encoding an enzyme according to any of embodiments 1-10.

[0246] 12. A recombinant expression vector comprising a DNA construct according to embodiment 11.

[0247] 13. A cell that has been transformed with a DNA construct of embodiment 11 or the vector of embodiment 12.

[0248] 14. A preparation comprising an enzyme according to any one of embodiments 1-10, or a DNA construct according to embodiment 11, or a vector according to embodiment 12, or a cell according to embodiment 13.

[0249] 15. A composition comprising an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to any one of embodiments 1, 2, 4-10 in combination with any one or more .beta.-glucanase.

[0250] 16. The composition according to embodiment 15, wherein said one or more .beta.-glucanase is an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to any one of embodiments 3-10.

[0251] 17. A composition comprising an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to any one of embodiments 3-10 in combination with any one or more xylanase.

[0252] 18. The composition according to embodiment 17, wherein said one or more xylanase is an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to any one of embodiments 1, 2, 4-10.

[0253] 19. The composition according to any one of embodiments 15-18, wherein said endo-1,3(4)-.beta.-glucanase activity and said endo-1,4-.beta.-xylanase activity are derived from at least two different enzymes, such as at least two different enzymes from two different species.

[0254] 20. The composition according to any one of embodiments 15-19, comprising a combination of at least two enzymes, said two enzymes, or two enzymes with an amino acid sequence having at least 80% sequence identity with the respective SEQ ID, or any functional fragment thereof, being selected from the list consisting of

SEQ ID NO:1 and SEQ ID NO:7;

SEQ ID NO:2 and SEQ ID NO:7;

SEQ ID NO:3 and SEQ ID NO:7;

SEQ ID NO:4 and SEQ ID NO:7;

SEQ ID NO:5 and SEQ ID NO:7;

SEQ ID NO:6 and SEQ ID NO:7;

SEQ ID NO:17 and SEQ ID NO:7;

SEQ ID NO:18 and SEQ ID NO:7;

SEQ ID NO:1 and SEQ ID NO:8;

SEQ ID NO:2 and SEQ ID NO:8;

SEQ ID NO:3 and SEQ ID NO:8;

SEQ ID NO:4 and SEQ ID NO:8;

SEQ ID NO:5 and SEQ ID NO:8;

SEQ ID NO:6 and SEQ ID NO:8;

SEQ ID NO:17 and SEQ ID NO:8;

SEQ ID NO:18 and SEQ ID NO:8;

SEQ ID NO:1 and SEQ ID NO:9;

SEQ ID NO:2 and SEQ ID NO:9;

SEQ ID NO:3 and SEQ ID NO:9;

SEQ ID NO:4 and SEQ ID NO:9;

SEQ ID NO:5 and SEQ ID NO:9;

SEQ ID NO:6 and SEQ ID NO:9;

SEQ ID NO:17 and SEQ ID NO:9;

SEQ ID NO:18 and SEQ ID NO:9;

SEQ ID NO:1 and SEQ ID NO:10;

SEQ ID NO:2 and SEQ ID NO:10;

SEQ ID NO:3 and SEQ ID NO:10;

SEQ ID NO:4 and SEQ ID NO:10;

SEQ ID NO:5 and SEQ ID NO:10;

SEQ ID NO:6 and SEQ ID NO:10;

SEQ ID NO:17 and SEQ ID NO:10;

SEQ ID NO:18 and SEQ ID NO:10;

SEQ ID NO:1 and SEQ ID NO:11;

SEQ ID NO:2 and SEQ ID NO:11;

SEQ ID NO:3 and SEQ ID NO:11;

SEQ ID NO:4 and SEQ ID NO:11;

SEQ ID NO:5 and SEQ ID NO:11;

SEQ ID NO:6 and SEQ ID NO:11;

SEQ ID NO:17 and SEQ ID NO:11;

SEQ ID NO:18 and SEQ ID NO:11;

SEQ ID NO:1 and SEQ ID NO:12;

SEQ ID NO:2 and SEQ ID NO:12;

SEQ ID NO:3 and SEQ ID NO:12;

SEQ ID NO:4 and SEQ ID NO:12;

SEQ ID NO:5 and SEQ ID NO:12;

SEQ ID NO:6 and SEQ ID NO:12;

SEQ ID NO:17 and SEQ ID NO:12;

SEQ ID NO:18 and SEQ ID NO:12;

SEQ ID NO:1 and SEQ ID NO:13;

SEQ ID NO:2 and SEQ ID NO:13;

SEQ ID NO:3 and SEQ ID NO:13;

SEQ ID NO:4 and SEQ ID NO:13;

SEQ ID NO:5 and SEQ ID NO:13;

SEQ ID NO:6 and SEQ ID NO:13;

SEQ ID NO:17 and SEQ ID NO:13;

SEQ ID NO:18 and SEQ ID NO:13;

SEQ ID NO:1 and SEQ ID NO:14;

SEQ ID NO:2 and SEQ ID NO:14;

SEQ ID NO:3 and SEQ ID NO:14;

SEQ ID NO:4 and SEQ ID NO:14;

SEQ ID NO:5 and SEQ ID NO:14;

SEQ ID NO:6 and SEQ ID NO:14;

SEQ ID NO:17 and SEQ ID NO:14;

SEQ ID NO:18 and SEQ ID NO:14;

SEQ ID NO:1 and SEQ ID NO:15;

SEQ ID NO:2 and SEQ ID NO:15;

SEQ ID NO:3 and SEQ ID NO:15;

SEQ ID NO:4 and SEQ ID NO:15;

SEQ ID NO:5 and SEQ ID NO:15;

SEQ ID NO:6 and SEQ ID NO:15;

SEQ ID NO:17 and SEQ ID NO:15;

SEQ ID NO:18 and SEQ ID NO:15;

SEQ ID NO:1 and SEQ ID NO:16;

SEQ ID NO:2 and SEQ ID NO:16;

SEQ ID NO:3 and SEQ ID NO:16;

SEQ ID NO:4 and SEQ ID NO:16;

SEQ ID NO:5 and SEQ ID NO:16;

SEQ ID NO:6 and SEQ ID NO:16;

SEQ ID NO:17 and SEQ ID NO:16; and

SEQ ID NO:18 and SEQ ID NO:16.

[0255] 21. The composition according to any one of embodiments 15-20, wherein when used prior to the lautering in a brewing application the total pressure built up is reduced to a value of less than 470 mm WC, such as less than 450 mm WC, such as less than 430 mm WC, such as less than 410 mm WC, such as less than 390 mm WC, such as less than 370 mm WC, such as less than 350 mm WC, such as less than 330 mm WC, such as less than 310 mm WC, such as less than 300 mm WC, such as less than 290 mm WC.

[0256] 22. The composition according to any one of embodiments 15-21, wherein when used prior to the lautering in a brewing application the total pressure built up is reduced by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95% compared to the use of a negative control without said composition.

[0257] 23. The composition according to any one of embodiments 15-22, wherein when used in a brewing application prior to the wort separation, the wort filterability as measured by volume wort collected after 5 min of filtration relative to a control without enzymes is increased to above 1.5, such as above 1.6, such as above 1.7, such as above 1.8, such as above 1.9, such as above 2.0, such as above 2.1, such as above 2.2, such as above 2.3, such as above 2.4, such as above 2.5.

[0258] 24. The composition according to any one of embodiments 15-23, when used in a brewing application prior to the wort separation, the wort filterability as measured by volume wort collected after 5 min of filtration is increased by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300% as compared to the use of a negative control without said composition.

[0259] 25. The composition according to any one of embodiments 15-24, comprising any one or more further enzyme.

[0260] 26. The composition according to embodiment 25, wherein said one or more further enzyme is selected from list consisting of a xylanase classified in EC 3.2.1.32, EC 3.2.1.136, or EC 3.2.1.156, a cellulase, a laminarinase, an endo-1,5-.alpha.-L-arabinanase, a beta-D-glucoside glucohydrolase, a .beta.-Xylosidase, a cellobiohydrolase, a glucan 1,4-beta-glucosidase, a xyloglucan-specific exo-beta-1,4-glucanase and an .alpha.-N-Arabinofuranosidase.

[0261] 27. Use of an enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26 in the production of a food, feed, or malt beverage product.

[0262] 28. Use of an enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26, in the production of dough or baked products.

[0263] 29. Use of an enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26, in the preparation of pulp or paper.

[0264] 30. Use of an enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26, for the preparation of cereal components.

[0265] 31. The use according to embodiment 29, in which the cereal is rye, wheat, or barley.

[0266] 32. Use of enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26, in the production of beer or modification of by-products from a brewing process.

[0267] 33. Use of enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26, in the production of wine or juice.

[0268] 34. Use of enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26, in the production of a first- or second-generation biofuel, such as bioethanol.

[0269] 35. Method of altering filterability of a starch comprising material, said method comprising the step of treating said starch comprising material with enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26.

[0270] 36. Method of reducing pressure built up during lautering in a brewing application, said method comprising the step of treating a brewing mash with enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26.

[0271] 37. Method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, said method comprising the step of treating a starch comprising material with enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26.

[0272] 38. Method for the production of a brewing mash, said method comprising the step of treating a starch comprising material with enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26.

[0273] 39. Method for the production of a first- or second-generation biofuel, such as bioethanol, said method comprising the step of treating a starch comprising material with an enzyme according to embodiments 1-10, or a preparation according to embodiment 14, or a composition according to any one of embodiments 15-26.

[0274] 40. Product obtained by the method according to any one of embodiments 38-39.

[0275] 41. A composition comprising the product according to embodiment 40, such as wherein the product is in a range of 0.1%-99.9%.

EXAMPLES

Example 1

[0276] Methods and results in relation to xylanases/glucanase filing for brew application.

[0277] The below methods have been used to screen for xylanases and glucanases with application in brewing:

[0278] Methods:

[0279] Water Extractable Arabinoxylan (WE-AX) Xylanase Method:

[0280] Samples, to obtain approx. OD540=0.25-0.30 in this assay and xylose standards (0, 0.125, 0.250, 0.375 and 0.500 mg/ml distilled water) are prepared in distilled water. At time t=0 minutes, 1.75 ml soluble wheat arabinoxylan (0.5% wheat arabinoxylan (PWAXYH, Megazyme, Bray, Ireland)) in 0.1M sodium acetate/acetic acid, pH 5) is placed in a test tube at 50.degree. C. At time t=5 minutes, 250 .mu.l enzyme solution is added to the substrate at 50.degree. C. followed by mixing. Distilled water is used as blank. At time t=15 minutes, 2 ml DNS solution (1% 3,5-Dinitrosalicylic acid (DNS), 1.6% sodium hydroxide, 30% potassium sodium tartrate in distilled water) is added to the enzyme-substrate solution and 2.0 ml standard solution. Samples, blanks and standards added DNS are placed in a boiling water bath (95.degree. C.) for 5 minutes. Hereafter samples, blanks and standards are cooled by placing them in a 25.degree. C. water bath for 20 minutes. The Optical density of all samples are read at OD540 using a spectrophotometer. Based on the dilution of the samples, the amount of sample taking into work and the standards, the xylanases activity of the sample can be calculated.

[0281] One Unit of endo-1,4-beta-xylanase WE-AX activity is defined as the amount of enzyme which produces 1 .mu.mole xylose equivalents per minute under the conditions mentioned above (Water extractable arabinoxylan (WE-AX) xylanase method).

[0282] Water Un-Extractable Arabinoxylan (WU-AX) Xylanase Method:

[0283] Samples are prepared in distilled water. At time t=0 minutes, 1.75 ml Insoluble wheat (0.5% wheat arabinoxylan (PWAXYI, Megazyme, Bray, Ireland)) in 0.1M sodium acetate/acetic acid, pH 5) is placed in a test tube at 50.degree. C. At time t=5 minutes, 250 .mu.l enzyme solution is added to the substrate at 50.degree. C. followed by mixing. Distilled water is used as blank. At time t=15 minutes, samples and blanks are placed in a boiling water bath (95.degree. C.) for 5 minutes.

[0284] Hereafter samples and blanks are centrifuged to precipitate residual insoluble substrate. The amount of arabinoxylan brough into solution is determined using the method described by Rouau, X. and Surget, A. (1994), Carbohydrate Polymers, 24, 123-132.

[0285] WU-AX endo-1,4-beta-xylanase activity is defined as the amount of pentoses solubilised (.mu.g pentoses) under the conditions described above giving a unit definition of .mu.g pentose/gram of xylanase sample.

[0286] Xylanase Activity Assay

[0287] Samples, to obtain approx. OD540=0.25-0.30 in this assay and xylose standards (0, 0.125, 0.250, 0.375 and 0.500 mg/ml distilled water) are prepared in distilled water. At time t=0 minutes, 1.75 ml soluble wheat arabinoxylan (0.5% wheat arabinoxylan (PWAXYH, Megazyme, Bray, Ireland)) in 0.1M sodium acetate/acetic acid, pH 5) is placed in a test tube at 50.degree. C. At time t=5 minutes, 250 .mu.l enzyme solution is added to the substrate at 50.degree. C. followed by mixing. Distilled water is used as blank. At time t=15 minutes, 2 ml DNS solution (1% 3,5-Dinitrosalicylic acid (DNS), 1.6% sodium hydroxide, 30% potassium sodium tartrate in distilled water) is added to the enzyme-substrate solution and 2.0 ml standard solution. Samples, blanks and standards added DNS are placed in a boiling water bath (95.degree. C.) for 5 minutes. Hereafter samples, blanks and standards are cooled by placing them in a 25.degree. C. water bath for 20 minutes. The Optical density of all samples are read at OD540 using a spectrophotometer. Based on the dilution of the samples, the amount of sample taking into work and the standards, the xylanases activity of the sample can be calculated.

[0288] One Unit of endo-1,4-beta-xylanase WE-AX activity is defined as the amount of enzyme which produces 1 .mu.mole xylose equivalents per minute under the conditions mentioned above

[0289] Glucanase Activity Assay

[0290] Samples, to obtain OD.sub.540 within the standard curve in this assay and glucose standards (0; 0.125; 0.250; 0.500; and 0.750 mg/ml distilled water) are prepared in distilled water. At time t=0 minutes, 1.75 ml barley beta-glucan (1.5% barley beta-glucan (P-BGBM, Megazyme, Bray, Ireland)) in 1M sodium acetate/acetic acid, pH 5) is placed in a test tube at 50.degree. C. At time t=5 minutes, 250 .mu.l enzyme solution is added to the substrate at 50.degree. C. followed by mixing. Distilled water is used as blank. At time t=15 minutes, 2 ml DNS solution (1% 3,5-Dinitrosalicylic acid (DNS), 1,6% sodium hydroxide, 30% potassium sodium tartrate in distilled water) is added to the enzyme-substrate solution and 2.0 ml standard solution. Samples, blanks and standards added DNS are placed in a boiling water bath (95.degree. C.) for 15 minutes. Hereafter samples, blanks and standards are cooled by placing them in a 25.degree. C. water bath for 20 minutes. The Optical density of all samples are read at OD.sub.540 using a spectrophotometer. Based on the dilution of the samples, the amount of sample taking into work and the standards, the glucanase activity of the sample can be calculated.

[0291] One unit of endo-1,3(4)-.beta.-glucanase activity is defined as the amount of enzyme which produces 1 .mu.mole glucose equivalents per minute under the conditions of the assay (pH 5.0 (or as specified) and 50.degree. C.).

[0292] Lab Scale Brewing Application Method:

[0293] Lab scale brewing application studies were conducted using Pilsner malt:Barley in a 75:25 ratio at a water:grist ratio of 3:1 (150 ml:50 g grist). Initially water was preheated to 53.degree. C. before mashing in and pH adjustment (5.4, 2 M H2SO4). After regaining initial temperature (10 min period) the mashing profile (see FIG. 1) is initiated and enzymes are added. After mashing off wort separation is conducted using a conventional plastic funnel and filter paper (paper filter No 1, 24 cm diameter, Whatman, England). Filtration performance was evaluated as well as several other wort parameters, such as i.e. viscosity, .beta.-glucan and pentosan.

[0294] Wort filtration was measured for 30 min after which filtration was terminated. Collected wort was cooled before any further analysis.

[0295] Filtration

[0296] Filtration data are presented as volume wort collected after 5, 10, 15 and 30 minutes relative to a blank (brewing without added exogenous enzymes).

[0297] Pilot Scale Brewing

[0298] Trials were conducted in a pilot scale brewing facility (2 HL capacity). Wort separation was conducted by lautering and beer filtration by horizontal kiselguhr filtration.

[0299] To elucidate filtration optimization by combination of glucanase and xylanase under "challenging" brewing conditions, pilot scale brewing trails were conducted using a mixed grist comprising of 75% malt and 25% barley. Initially, the water:grist ratio was set at 2.8:1 (mash start) increasing to 3.1:1 at the start of lautering. In comparison water:grist ratios around 3.2-3.8 are typical in full scale brew house lautering. Thus the current pilot trial settings of a 3.1:1 water:grist ratio are believed to be in the challenging end of the scale.

[0300] Malt and barley was ground dry using a two-roller mill. Both barley and malt was milled twice using a roller distance of .about.0.7 mm.

[0301] Mashing-in was conducted aiming at an initial mash temperature of 53.degree. C. After mashing-in small adjustments were conducted such as: mash volume adjustment for water:grist ratio of 2.8:1 and pH adjustment to .about.5.56 (Lactic acid). After fine tuning the mash, enzyme was added and the mashing profile given in FIG. 1 was followed. Saccharification rest at 70.degree. C. was programmed to 15 min, however rest period was extended by 5 min until an iodine test showed that no starch was present. (Ludwig Narziss and Werner Back, Technische Universitaet Muenchen (Fakultaet fuer Brauwesen, Weihenstephan), Abriss der Bierbrauerei. WILEY-VCH Verlags GmbH Weinheim Germany, 2005).

[0302] Mashing-off was initiated after a 5 min rest at 78.degree. C. Mash was transferred to the Lauter Tun, which was beforehand prefilled with water to a height just below the "false bottom". The mash was left to rest for 5 min for settling of filter cake. This was followed by a 15 min recirculation (140 L/h) ensuring filter cake settling and wort clarification. Typically in full scale brewing, filtration will be initiated when a given wort turbidity is obtained, however in the current trials recirculation was kept constant at 15 min enabling comparison of trials. During lautering the following data were collected, including time (min), wort volume collected (L), filtration pressure difference across filter cake (mmWC, mm Water Column), pump capacity (%), wort turbidity (EBC) and mash temperature (.degree. C.).

[0303] The pressure build up across the filter cake during filtration is believed to be a factor contributing to setting the standard of the wort lautering performance. Reaching very high pressure differences--e.g. 250 mmWC during first wort collection and e.g. 450 mmWC for the reminder of the lautering--a filter cake racking (also known as deep cut) is induced. Racking is a process where a filter cake collapses or a filtration channel formation is relieved by slowing cutting the filter cake with special designed knives. Following filter cake racking a 6 min wort recirculation (flow rate: 120 l/h) was introduced priming the filter cake for continued filtration. Filter cake racking relieves an otherwise compromised filtration performance which would otherwise also result in poor wort quality. If no pressure induced racking has been introduced by the beginning of the 3rd sparging, automatic rackings were conducted at the beginning of the 3rd and 4th spargings to ensure that no full filtration block would occur just before finishing wort separation.

[0304] Lautering was conducted with the settings illustrated in table 1.

TABLE-US-00004 TABLE 1 Lautering settings. Volumes collected (L), filtration flow (L/h) and Sparging volumes (L). Volume Filtration Sparging Wort collected, L flow, L/h volume, L First wort 0-60 130 1 st sparging 60-78 140 18 2nd sparging 78-96 160 18 3rd sparging 96-114 180 18 4th sparging 114-140 180 26

[0305] After end lautering, sweet wort was returned to the Mash Tun, heated to boiling and hops were added. Hopping was continued for 80 min and at the end of hopping pH is adjusting to 5.10.+-.0.05. Hops were cleared from the bitter wort by use of whirlpool and following wort was cooled to .about.8.degree. C. For fermentation, a bottom fermenting dried yeast (Saccharomyces cerevisiae) W34/70 from Fermentis was chosen. Yeast was rehydrated for 30 min and pitched at 100 g/HL. Main fermentation was hold for 5-6 days at 10.degree. C., followed by maturation at 15.degree. C. until attenuated and Diacetyl below 80 ppb. Beer was stored for another 2-3 weeks at 1.degree. C. and 0.7 bar before filtering.

[0306] Beer was filtered horizontally by use of 1.2 .mu.m PP-candle plates and kieselguhr. Up to 8 plates could be included in the filtration unit, resulting in a total filtration area of .about.0.5 m2. In the current studies 3 plates were included and filtration was conducted at a flow rate of 130 L/h, resulting in a speed of filtration of 6.9 HL/(hm2). In full scale breweries, speed of filtration is usually set between 5-7 HL/(hm2). It is thus obvious that the current settings are in the high end--a deliberate choice challenging the beer filtration conditions to verify potential benefits from the choice of using an enzyme in the brewing process. During beer filtration, flow rates (L/h) as well as pressure values (P-in and P-out) were monitored to verify beer filtration performance. Also a number of beer analyses, such as Original Gravity (OG), Apparent Extract (AE), Alcohol By Volume (ABV), Apparent Degree of Fermentation (ADF), Reel Degree of Fermentation (RDF), pH, colour and bitterness were conducted for evaluation of beer quality.

[0307] Results:

[0308] Xylanases:

[0309] Xylanases were screened for their activity on soluble substrate and insoluble substrate, their pH and temperature characteristics.

[0310] Results are shown in table 2.

TABLE-US-00005 TABLE 2 Xylanases screened, their activity on soluble (WE-AX) and insoluble (WU-AX) arabinoxylan substrate and their biochemical characteristics in regard to temp and pH. Temp T1/2 WU-AX/ opt, temp, pH Name Origin GH WE-AX WU-AX WE-AX .degree. C. .degree. C. opt. AfuXyn2 Aspergillus 11 7798 68790526 8822 65 59 5.5 fumingatus AfuXyn3 Aspergillus 11 26283 99716865 3794 60 62 5 fumingatus AfuXyn5 Aspergillus 11 90005 714363158 7937 60 50 4 fumingatus BsuXyn3 Bacillus 11 82 1095357 13388 50 n.d. 6 subtilis, BS3 BsuXyn4 Bacillus 11 54 1005400 18619 50 n.d. 6 subtilis, BS4 #160 TerXyn1 Geosmithia 10 1467 6208786 4232 78 >78 3 emersonii AtuXyn3 Aspergillus 10 1220 7760982 6361 65 67 4.5 tubigensis AtuXyn4 Aspergillus 11 1600 12934971 8084 45 58 5 tubigensis AacXyn2 Aspergillus 10 777 3880491 4994 70 73 4 aculeatus TreXyn2 Trichoderma 11 2244 16015846 7137 55 n.d. 5 reesei TreXyn3 Trichoderma 10 21487 141108772 6567 60 64 5.5 reesei TreXyn5 Trichoderma 11 1410 8842816 6272 70 68 5 reesei n.d. = Not determined

[0311] WE-AX and WU-AX enzyme activities (U) were measured as described in sections "water extractable arabinoxylan (WE-AX) xylanase method" and "water un-extractable arabinoxylan (WU-AX) xylanase method".

[0312] Based on the results from the biochemical screening, xylanases having an appropriate activity ratio on soluble vs. insoluble arabinoxylan were chosen for further testing in application trials. The results are shown in table 3.

TABLE-US-00006 TABLE 3 Xylanases screened and the relative extract yield obtained using the xylanases versus a blank (without xylanases). Finally the xylanases substrate specificity is illustrated as a ration of their activity on insoluble vs. soluble arabinoxylan (WU-AX/WE-AX). Filtration Performance 5 10 15 30 WU-AX/ Name Origin min min min min WE-AX Blank 1.00 1.00 1.00 1.00 BsuXyn3 Bacillus 0.93 0.95 0.96 0.95 13388 subtilis, BS3 BsuXyn4 Bacillus n.d. n.d. n.d. n.d. 18619 subtilis, BS4 #160 TerXyn1 Geosmithia 2.19 1.92 1.70 1.44 4232 emersonii (Taleromyces emersonii) AtuXyn3 Aspergillus 2.06 1.75 1.59 1.37 6361 tubigensis AtuXyn4 Aspergillus 1.02 1.01 1.01 1.01 8084 tubigensis AacXyn2 Aspergillus 2.07 1.86 1.67 1.43 4994 aculeatus TreXyn3 Trichoderma 2.41 2.02 1.81 1.55 6567 reesei TreXyn5 Trichoderma 2.06 1.75 1.59 1.37 6272 reesei

[0313] Filtration performance was measured as described earlier ("filtration"), and is presented as volume filtrate at the different time points relative to the negative control (blank).

[0314] WE-AX and WU-AX enzyme activities (U) were measured as described in sections "water extractable arabinoxylan (WE-AX) xylanase method" and "water un-extractable arabinoxylan (WU-AX) xylanase method".

[0315] Glucanases:

[0316] Glucanases were screened for their activity and temperature characteristics, and the results are shown in table 4.

TABLE-US-00007 TABLE 4 Glucanases screened, their activity and their biochemical characteristics in regard to temperature Temp T1/2 T1/2 opt, temp_buffer, temp_wort, pH Name Origin U/ml .degree. C. .degree. C. .degree. C. opt. TerGlu1 Talaromyces 7338 70 78 78 3 emersonii/ Geosmithia emersonii BsuGluS Bacillus 208 55-65 60 68 .sup. 5-6 subtilis BsuGlu103FULL Bacillus 391 50-60 53 58 .sup. 5-6 subtilis TreGlu2 Trichoderma 13 40-50 70 74 4.5-6 reesei TreGlu3 Trichoderma 9215 40-51 58 62 4.5-6 reesei TreGlu4 Trichoderma n.d. 40-52 62 62 4.5-6 reesei TreGlu6 Trichoderma n.d. 40-53 62 64 4.5-6 reesei TreGlu7 Trichoderma n.d. 40-54 62 62 4.5-6 reesei TreGlu8 Trichoderma n.d. 40-55 61 63 4.5-6 reesei BsuGluC CBD Bacillus 10 50-60 60 67 .sup. 5-6 subtilis n.d. = Not determined

[0317] Glucanase activity/units was/were determined as described in the glucanase activity assay as described above.

[0318] Based on the results from the biochemical screening, glucanases having suitable characteristics were chosen for further testing in application trials. The results are shown in table 5.

TABLE-US-00008 TABLE 5 Name and origin of glucanases screened and the relative extract yield obtained using the glucanases versus a blank (without enzyme). Filtration performance Name Origin 5 min 10 min 15 min 30 min Blank Neg control 1.00 1.00 1.00 1.00 TerGlu1 Geosmithia 1.36 1.43 1.46 1.36 emersonii BsuGluS Bacillus 1.48 1.49 1.48 1.35 subtilis BsuGlu103FULL Bacillus 1.29 1.28 1.30 1.22 subtilis TreGlu2 Trichoderma 1.15 1.18 1.20 1.15 reesei TreGlu3 Trichoderma 1.29 1.32 1.30 1.22 reesei TreGlu4 Trichoderma 1.11 1.11 1.11 1.09 reesei TreGlu6 Trichoderma 1.13 1.15 1.13 1.10 reesei TreGlu7 Trichoderma 1.06 n.d. 1.01 1.02 reesei TreGlu8 Trichoderma 1.12 1.11 1.13 1.09 reesei BsuGluC CBD Bacillus 1.33 1.37 1.37 1.32 subtilis

[0319] Filtration performance was measured as described earlier ("filtration"), and is presented as volume filtrate at the different time points relative to the negative control (blank).

[0320] Based on the individual screening of xylanases and glucanases, combinatorial experiments were conducted, and results are illustrated in table 6.

TABLE-US-00009 TABLE 6 Brewing application results from combinatorial experiments of xylanases and glucanases versus a blank and versus UltraFlo .RTM. Max. 250 Fungal Xylanase Units FXU- S/g; 700 Cellulase Units EGU/g (Novozymes, Denmark) results are illustrated as relative extract yield obtained. Filtration Performance Name Origin 5 min 10 min 15 min 30 min Control 1.00 1.00 1.00 1.00 UFmax 0.1 A. aculeatus 2.29 2.13 2.00 1.77 BsuGluS/ B. sub/ 1.70 1.69 1.00 1.47 TauXyn1 T. aurantiacus BsuGluS/ B. sub/ 2.57 2.14 1.96 1.75 AtuXyn3 A. tubingensis

[0321] (Origin of UltraFlo.RTM. Max may include other microorganisms than A. aculeatus, such as described in WO05059084)

[0322] Filtration performance was measured as described earlier ("filtration"), and is presented as volume filtrate at the different time points relative to the negative control (blank).

[0323] Suitable combinations were further tested in a 2HL pilot scale facility for verification, and results are shown in table 7 and FIG. 3.

TABLE-US-00010 TABLE 7 Pilot scale Brewing application results from verification of the glucanase and xylanases screening. The B. sub glucanase S combined with the A. tub xylanases were tested against a blank and UltraFlo .RTM. Max. Data collected was the average flow (L/h), the total pressure build up over the lautering (mm WC) and the max pressure recorded during the lautering (mm WC). Avg Flow Total pressure Max pressure ID (L/h) build up (mm WC) (mm WC) Blank 148 556 356 UltraFlo max 149 478 280 BsuGluS/ 147 263 163 AtuXyn3

Example 2

[0324] In this example it was attempted to show that xylanases for brewing applications may have a very high selectivity for High Molecular Weight Soluble-arabinoxylan (HMWS-AX) and water extractable arabinoxylan (WE-AX). It is believed that hereby only limited amounts of arabinoxylan need to be solubilised. Consequently, the related off flavour potential is highly reduced.

[0325] A significantly reduced viscosity is facilitating mash and beer separation. Desired xylanase characteristics for brewing applications may include one or more of the following aspects of table 8:

TABLE-US-00011 TABLE 8 Screening criterias for xylanase selection Enzyme substrate specificity WE-AX/WU-AX ratio has an impact on viscosity Enzyme substrate selectivity How close to branch points the enzyme will cut has an impact on the functionality Enzyme thermostability Continuous solubilisation of AX during mashing - thermostability is a key feature Enzyme pH optimum (pH 5.4-5.6) Enzyme inhibition (e.g. known key factor for xylanases)

TABLE-US-00012 TABLE 9 Xylanases - biochem characteristics Inhibition by endogenous cereal xylanase inhibitors occur in both xylanase GH's Xylanase GH GH10 GH11 Mw +30 kDa 20 kDa Substrate Hydrolyse close Need more specificity to Arabinose unsubstituted substitutions Xylose to hydrolyse AX Substrate WE-AX/WU-AX WE-AX/WU-AX selectivity typically >1 typically <1 SBD Often separate No classical SBD SBD, but secondary BD on surface Technological Viscosity Solubilizer/ effect reducers viscosity reducers

[0326] Water-Unsoluble ArabinoXylan (WU-AX) in cereals as shown in FIG. 3 is linked to filter cake stability in the brew house.

[0327] The concentration of ferulic acid (FA) in cereals very much depends on the tissue. The highest concentration is found in the pericarp material, whereas the concentration in the endosperm is much lower. Different concentrations are reported. A concentration of 2700 .mu.g/g insoluble fiber, 185 .mu.g/g soluble fiber is likely (Bunzel et al. 2001, Journal of Sc. of food and agriculture, vol. 81, p. 653-60).

[0328] To put this into perspective it means that FA is only found for every 200th xylose molecules in arabinoxylan in insoluble fiber (WU-AX) and for every 2500 xylose in soluble fiber (WE-AX).

[0329] It is a well-known fact that xylanases may lead to off-flavor formation in beer such as free ferulic acid and 4-VG.

[0330] Methods:

[0331] Based on the criteria mentioned in table 8+9 more than 15 xylanases from DuPont Industrial Biosciences were found as potential candidates. The xylanases were screened in laboratory mashing application applying up to 30% barley in combination with malt. Among others, mash separation speed, pentosan/arabinoxylan level and wort viscosities were monitored. Top candidates where tested at several pilot brewery plant studies to test our hypothesis and link xylanase characteristics to functionality in brewing. The optimal dosage of the selected xylanase candidate was tested in combination with a .beta.-glucanase.

[0332] Results and Discussion:

TABLE-US-00013 TABLE 10 Sample ID Ref Control (X + B). X1 X2 X3 Dyn. 1.798 1.670 1.801 1.746 1.794 Viscosity (12.degree. Plato) mPa s Extract 15.1 15.7 15.6 15.2 15.1 (.degree. Plato) Total 1610 1910 2440 2020 1710 pentosan (mg/l)

[0333] Pilot plant brews where enzyme dosage is the only variable. Applying a WU-AX selective xylanase (X1) results in filter bed collapse. WE-AX selective xylanase candidates (reference, X2, X3) results in low pressure buildup. The reference is a blend of xylanase+beta-glucanase.

TABLE-US-00014 TABLE 11 Wort analyses - pilot plant studies Sample ID Ref. X + B Xh + B Extract (.degree. Plato) 15.70 16.00 15.95 Betaglucan in wort (mg/l) 44 35 25 Dyn. Viscosity at 1.65 1.68 1.68 12.degree. Plato (mPa s) Total pentosan (mg/l) 3540 2970 3010

TABLE-US-00015 TABLE 12 Strecker Aldehyd analysis of aged beer Aging markers (forced aged beer) Unit Ref. X + B Xh + B 2-Me--Pr ppb 25 24 22 2-Me--Bu ppb 3 2 3 3-Me--Bu ppb 9 7 8 Furfural ppb 113 85 93 Methional ppb 6 5 6 PheAcal ppb 10 9 10 T2N ppb 0.022 0.022 0.022

[0334] Optimized blends of a WE-AX selective xylanase applied at a medium (X) and a high dosage (Xh) in combination with .beta.-glucanase (B) on 20% barley/80% malt. The results indicate a good mash and beer separation performance with a low risk of off-flavor formation and filter bed collapse.

[0335] Conclusion

[0336] The study has proven the importance of applying xylanases for brewing which are highly selective for the WE-AX during mashing. The following benefits are achieved: [0337] Good mash separation and beer filtration performance [0338] Minimized risk of filter bed collapse at lautering [0339] Reduced potential for off-flavor formation related to arabinoxylan breakdown [0340] Tolerance towards xylanase overdose

[0341] Xylanases can often be applied with a high beneficial effect in combination with beta-glucanases for separation control.

Example 3

[0342] Evaluation of X3/BglS (also referred to as AtuXyn3/BsuGluS) combinations in 2 HL Pilot brewing trials

[0343] Material and Methods:

[0344] Experiments: Enzymes:

[0345] AtuXyn3 (X3)/BsuGluS (Bgls) (a): Combination of BglS (Bacillus glucanase) and X3 (Aspergillus xylanase; BgLS: 0.50 mg protein/kg grist and X3: 1.50 mg protein/kg grist).

[0346] AtuXyn3 (X3)/BsuGluS (Bgls) (b): As AtuXyn3 (X3)/BsuGluS (Bgls) (a), but with 20% increase X3 dose to test robustness.

[0347] Reference: Benchmark enzyme product (Ultraflo.RTM. Max) dosed at 0.20 kg/T grist.

[0348] Raw Material:

[0349] Adjunct material: barley 22% w/w.

[0350] Malt: Pilsner malt Chiraz 42.6% w/w, Pilsner malt Quench DMG 35.4% weight pr. weight (ww).

[0351] All material used for acid adjustment of pH, Calcium, Zink and bitterness levels are food grade and considered as standard brewing materials.

[0352] The recipe for the brew was aiming at a beer style as an international lager beer.

[0353] Milling:

[0354] Kunzel 2 roller pilot mill. The milled material was passing the rollers twice simulating a 4 roller mill.

[0355] Malt grist: the mill was running at 1.5 mm at the first pass and 0.7 mm at the second pass of the rollers.

[0356] Barley grist: the mill was running at 1.5 mm at the first pass and 0.4 mm at the second pass of the rollers.

[0357] Brewhouse 2 HL:

[0358] All brews were based on HGB (High Gravity Brewing) infusion mashing and standard lautering of 190 L wort aiming at 16.degree. Plato. During lautering which was performed at fixed flow, the differential pressure was recorded (used as parameter for evaluating lautering performance). All brew materials were milled ahead of time (24 h) and kept in closed buckets prior to water contact. All material was dumped in the mash kettle within the first 3 minutes after start of mashing. Calcium and pH adjustment were done prior to enzyme addition. pH (20.degree. C.) was rechecked at the 52.degree. C. break. Iodine normality was confirmed after 10 minutes at 72.degree. C. Lautering was performed at 78.degree. C.

[0359] Lautering performance was evaluated on fixed flow at 90 l/H during first wort collection. Flow was increased to 110 L/hour and 130 L/hour during sparging and weak wort collection. Chemical analysis was performed on cold wort.

[0360] Wort Boiling:

[0361] Boiling was performed using an external boiler with 4-5% evaporation. Hop extracts were added from the beginning of the wort boiling aiming at 20 BU in the final beer.

[0362] Fermentation 50 L:

[0363] All fermentations were performed in 50 L cylindriconical tanks. Fermentation was made according to standard operation procedures. Pitching was done with 15.times.10.sup.6 live yeast cells/ml. Yeast counts and viability was calculated using a Nucleo counter.

[0364] Beer Processing:

[0365] Plate and frame filter operated at constant pressure. Flow evaluation was done by weight.

[0366] Data was collected from 1 and 3 filter plates.

[0367] Debrewing:

[0368] All beers were de-brewed to 5.0% ABV (Alcohol By Volume), considered as international lager beer standard.

[0369] Bottling:

[0370] CO.sub.2 was adjusted to 5.0 g/L. All beer samples were bottled in 33 cl standard bottles on a McLennon automatic filling machine using single evacuation.

[0371] Beer Analysis:

[0372] Fresh beers were analysed using GC-MS

[0373] Chemical aging profile was determined using GC-MS.

[0374] Results and observations: Mashing.

[0375] Mashing was performed with the following condition:

[0376] 52.degree. C. for 10 minutes simulating a 15-20 minutes mashing using a running mill.

[0377] 65.degree. C. for 40 minutes.

[0378] 72.degree. C. for 30 minutes.

[0379] 78.degree. C. for 10 minutes.

[0380] All ramping steps were executed at 1.degree. C. A graphic representation is given in FIG. 7.

[0381] All trials were made with this mashing regime aiming at a 16.degree. Plato brew. There were no remarks to this process step.

[0382] Results and observations: Lautering.

[0383] The lautering was performed in the 2 hl brewery with a load of 150 kg/m.sup.2. This is representative for a standard brew house operation. Control of the lautering process was made as a fixed flow at an average of 100 litre/hour. Initial flow rate is 90 litre/hour, increasing to 130 litre/hour during weak wort collection. Differential pressure and in-line measurement of haze was recorded for the four brews. Total lautering and wort collection was done over approximately 2 hours.

[0384] Trial X3/BglS (b) and X3/BglS (a) are suggested to be the trials that had the best lautering performance followed by trial X3/BglS (a) and trial UF max with the worst performance.

TABLE-US-00016 TABLE 13 Data collected during lautering of the mash from the four trials. UF X3/BglS X3/BglS X3/BglS max (a) (b) (a) Lauter tun load (kg/m3) 153 153 153 153 Lauter tun time (min) 154 164 170 154 Diff. Pressure (cm) 40 30 30 30 Racking (# deep cuts) 1 1 1 1 Haze (EBC) 10 15 10 10 First wort pressure 40 33 31 30 build up (cm/h) Time to first deep cut 45 60 120 115 (min) "Diff. Pressure" and "First wort pressure build up" in the table was measured as cmWC (cm Water column) and not as (cm) and (cm/h) respectively.

[0385] Results and observations: Wort analysis after boiling.

[0386] Analysis of the cold wort shows similar results. The beta-glucan analysis indicates a slight difference between the samples.

TABLE-US-00017 TABLE 14 Chemical analysis of the cold wort. UF X3/BglS X3/BglS X3/BglS Wort max (a) (b) (a) Extract 16.09 16.05 15.99 16.1 (% plato) Color (EBC) 9.7 9.3 9.3 9.3 pH 5 5.2 5.2 5.2 Iodine (Y/N) N N N N Bitterness 52 51 46 50 (BU, EBC)

TABLE-US-00018 TABLE 15 Analytical data on cold wort. UF X3/BglS X3/BglS X3/BglS max (a) (b) (a) Beta-glucan in wort 49 40 25 30 (mg/L) Dyn. Viscosity at 1,888 1,685 1,679 1,686 12.degree. C. (mPa s) Pentosan (mg/l) 3365 2975 3014 2964 Ferulic acid (ug/ml) 4.3 3.9 3.8 3.9 4-VG (ug/ml) <0.49 <0.49 <0.49 <0.49 (12.degree. C. is 12.degree. Plato); (% Plato may be used interchangeably with .degree. Plato)

[0387] Results and observations: Fermentation.

[0388] Analysis of the green beer is given in table 16.

TABLE-US-00019 TABLE 16 Green beer analysis. UF X3/BglS X3/BglS X3/BglS Green beer max (a) (b) (a) Alcohol (% vol) 6.79 6.79 6.7 6.86 Real extract (% P) 6.28 6 6 6 RDF (%) 63.5 63.7 63.5 64.4 Original extract 16.29 16.25 16.09 16.24 (% P) Color (EBC) 8.3 8.5 -- -- pH 4.4 4.4 4.4 4.4 SO2 (ppm) 7 9 11 10 Bitterness (BU, EBC) 29 28 27 27

[0389] The green beer analysis shows a high degree of similarity between the trials. All trials have relative low RDF, but this is normally seen with the inclusion of 22% barley calculated on the basis of weight per weight (ww).

[0390] Results and observations: Beer filtration.

[0391] Beer samples were filtered using a plate and frame filter using a fixed pressure. Two kegs of approximately 15 kg were filtered and the individual keg filtration data are presented in table 17. The first keg was filtered using 1 filter sheet and the second keg was filtered using 3 filter sheets. The differential pressure was always 0.5 bar. The filter plates are KD7 (20 cm.times.20 cm) from Begerow.

[0392] The overall picture of the filtrations curves from either 1 or 3 filter plate filtrations is the same. We believe that the 1 filter plate record may be too sensitive to show the real ratio difference.

TABLE-US-00020 TABLE 17 Keg filtration data from the four trials UF X3/BglS X3/BglS X3/BglS Filtration max (a) (b) (a) Filtration speed - 4.8 5.6 9.9 11.8 1 filter sheet (L/h) Filtration speed - 77.2 59.6 70.6 105.4 3 filter sheet (L/h) Results and observations: Final beer analysis.

[0393] Trial beers were analysed according to standard operation procedures (EBC) and presented in table 18.

TABLE-US-00021 TABLE 18 Final beer analysis. UF X3/BglS X3/BglS X3/BglS Finished beer max (a) (b) (a) alcohol (%) 4.82 4.89 5.01 4.92 Real extract (% P) 4.5 4.6 4.6 4.4 RDF (%) 63.2 63.4 63.8 64.3 Original extract 11.85 11.99 12.19 11.89 (% P) Color (EBC) 4.8 4.9 5 5 pH 4.4 4.4 4.4 4.4 SO2 (ppm) 13 13 10 6 Bitterness (BU, 22 22 20 18 EBC) Haze (EEC) 0.43 0.4 0.38 0.4 Total haze - 8.6 12.9 7.3 6.2 5 d-60 dg C. (EBC) CO2 (g/L) 4.9 5.3 5.1 5.2 Diacetyl (ppb) 12 11 8 10 Head retention (S) 107 111 119 108 Foam volume (ml) 452 476 460 470

[0394] Results and observations: Strecker aldehydes and "age markers" in final beer.

[0395] Analysis was performed both on fresh and aged beer. Strecker aldehydes and the "age and heat markers" (2-Me-Pr (2-methyl Propanal), 2-Me-Bu (2-methyl Butanal), 3-Me-Bu (3-methyl Butanal), Furfural, Methional, PheAcal (phenyl Acetaldehyde) and T2N (trans-2-nonenal)) were analysed by GC-MS on both fresh and aged beer. The data from analysis of fresh beer is presented in table 19.

TABLE-US-00022 TABLE 19 Strecker aldehyde analysis of fresh beer. Markers for heat and aging (Furfural and trans-2-Nonenal) are used as sample control. Aging markers UF X3/BglS X3/BglS X3/BglS (fresh beer) max (a) (b) (a) 2-ME--Pr (ppb) 5 5 5 6 2-ME--Bu (ppb) 2 2 2 2 3-ME--Bu (ppb) 6 6 6 6 Furfural (ppb) 10 11 11 10 Methional (ppb) 4 4 4 4 PheAcal (ppb) 6 6 6 7 T2N (ppb) 0.0011 0.005 0.004 0.006

[0396] The trial beers were incubated at 37.degree. C. for 2 weeks prior to the Strecker aldehyde analysis. The data for aged beer samples are presented in table 20.

TABLE-US-00023 TABLE 20 Strecker aldehyde analysis of aged beer. Markers for heat and aging (furfural and trans-2-Nonenal) is used as a sample control. Aging markers UF X3/BglS X3/BglS X3/BglS (forced aged beer) max (a) (b) (a) 2-ME--Pr (ppb) 25 23 22 26 2-ME--Bu (ppb) 3 3 3 2 3-ME--Bu (ppb) 9 7 7 8 Furfural (ppb) 111 92 93 78 Methional (ppb) 6 5 6 6 PheAcal (ppb) 10 9 9 10 T2N (ppb) 0.017 0.022 0.022 0.022

[0397] The data presented in table 20 show an expected increase in Strecker aldehyde level. The increase in furfural and trans-2-Nonenal reach an expected level.

[0398] Conclusion:

[0399] Based on the pilot scale experiments, we can conclude that the ratios of the BglS and X3 tested in this Experiment performs as good or even better than the reference UltraFlo Max in pilot scale brewing.

[0400] The results are surprising, seen in the light of the challenging raw material used, 22% barley inclusion in combination with the 300 mg/I 6-glucan containing malt. The performance is not only seen in the mash separation results, also in the beer filtration. Due to the low solubilisation of cell wall material when using the BREW2 (pentosan data), a lower degree of cell wall material that might cause quality issues in relation to off-taste and stability, can be recorded.

[0401] Finally it can be concluded that a 20% increase in the dose of the xylanase component in X3/Bgls (b) appears not to have any impact on any of the evaluated parameters, indicating that X3/Bgls (a) is a robust enzyme combination.

TABLE-US-00024 Sequences: AtuXyn3, Aspergillus tubigensis,302 aa (SEQ ID NO: 1) QASVSIDTKFKAHGKKYLGNIGDQYTLTKNSKTPAIIKADFGALTPENSMKWDATEPSRGQFSFSGS DYLVNFAQSNNKLIRGHTLVWHSQLPSWVQAITDKNTLIEVMKNHITTVMQHYKGKIYAWDVVNEIF NEDGSLRDSVFYQVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKLTGMVSHVKKWIEAGIP IDGIGSQTHLSAGGGAGISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLDQPKCIGITVW GVADPDSWRSSSTPLLFDSNYNPKPAYTAIANAL TerXynl, Geosmithia emersonii (Taleromyces emersonii) (SEQ ID NO: 2) AGLNTAAKAIGLKYFGTATDNPELSDTAYETQLNNTQDFGQLTPANSMKWDATEPEQNVFTFSAGDQ IANLAKANGQMLRCHNLVWYNQLPSWVTSGSWTNETLLAAMKNHITNVVTHYKGQCYAWDVVNEALN DDGTYRSNVFYQYIGEAYIPIAFATAAAADPNAKLYYNDYNIEYPGAKATAAQNLVKLVQSYGARID GVGLQSHFIVGETPSTSSQQQNMAAFTALGVEVAITELDIRMQLPETEALLTQQATDYQSTVQACAN TKGCVGITVWDWTDKYSWVPSTFSGYGDACPWDANYQKKPAYEGILTGLGQTVISTTYIISPTTSVG IGTTTSSGGSGGTTGVAQHWEQCGGLGWTGPTVCASGYTCTVINEYYSQCL AtuXyn4, Aspergillus tubigensis (SEQ ID NO: 3) EPIEPRQASVSIDTKFKAHGKKYLGNIGDQYTLTKNSKTPAIIKADFGALTPENSMKWDATEPSRGQF SFSGSDYLVNFAQSNNKLIRGHTLVWHSQLPSWVQSITDKNTLIEVMKNHITTVMQHYKGKIYAWDVV NEIFNEDGSLRDSVFYKVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKLTGMVSHVKKWIAA GIPIDGIGSQTHLSAGGGAGISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLNQPKCIGIT VWGVADPDSWRSSSTPLLFDSNYNPKPAYTAIANAL AacXyn2, Aspergillus aculeatus (SEQ ID NO: 4) MVGLLSITAALAATVLPNIVSAVGLDQAAVAKGLQYFGTATDNPELTDIPYVTQLNNTADFGQITPGN SMKWDATEPSQGTFTFTKGDVIADLAEGNGQYLRCHTLVWYNQLPSWVTSGTWTNATLTAALKNHITN VVSHYKGKCLHWDVVNEALNDDGTYRTNIFYTTIGEAYIPIAFAAAAAADPDAKLFYNDYNLEYGGAK AASARAIVQLVKNAGAKIDGVGLQAHFSVGTVPSTSSLVSVLQSFTALGVEVAYTEADVRILLPTTAT TLAQQSSDFQALVQSCVQTTGCVGFTIWDWTDKYSWVPSTFSGYGAALPWDENLVKKPAYNGLLAGMG VTVTTTTTTTTATATGKTFTTFTGATSTGTTAAHWGQCGGLNWSGPTACATGYTCTYVNDYYSQCL TreXyn3, Trichoderma reesei (SEQ ID NO: 5) MKANVILCLLAPLVAALPTETIHLDPELAALRANLTERTADLWDRQASQSIDQLIKRKGKLYFGT ATDRGLLQREKNAAIIQADLGQVTPENSMKWQSLENNQGQLNWGDADYLVNFAQQNGKSIRGHTL IWHSQLPAWVNNINNADTLRQVIRTHVSTVVGRYKGKIRAWDVVNEIFNEDGILRSSVFSRLLGE EFVSIAFRAARDADPSARLYINDYNLDRANYGKVNGLKTYVSKWISQGVPIDGIGSQSHLSGGGG SGTLGALQQLATVPVTELAITELDIQGAPTTDYTQVVQACLSVSKCVGITVWGISDKDSWRASTN PLLFDANFNPKPAYNSIVGILQ TreXyn5, Trichoderma reesei (SEQ ID NO: 6) QCIQPGTGYNNGYFYSYWNDGHGGVTYCNGPGGQFSVNWSNSGNFVGGKGWQPGTKNRVINFSGSY NPNGNSYLSVYGWSRNPLIEYYIVENFGTYNPSTGATKLGEVTSDGSVYDIYRTQRVNQPSIIGTA TFYQYWSVRRNHRSSGSVNTANHFNAWAQQGLTLGTMDYQIVAVEGYFSSGSASITVSD BsuGluS, Bacillus subtilis, 214 aa (SEQ ID NO: 7) QTGGSFFDPFNGYNSGFWQKADGYSNGNMFNCTWRANNVSMTSLGEMRLALTSPAYNKFDCGENRSV QTYGYGLYEVRMKPAKNTGIVSSFFTYTGPTDGTPWDEIDIEFLGKDTTKVQFNYYTNGAGNHEKIV DLGFDAANAYHTYAFDWQPNSIKWYVDGQLKHTATNQIPTTPGKIMMNLWNGTGVDEWLGSYNGVNP LYAHYDWVRYTKK TerGlu1, Geosmithia emersonii (Taleromyces emersonii) (SEQ ID NO : 8) APVKEKGIKKRASPFQWFGSNESGAEFGNNNIPGVEGTDYTFPNTSAIQILIDQGMNIFRVPFLMER MVPNQMTGPVDSAYFQGYSQVINYITSHGASAVIDPHNFGRYYNNIISSPSDFQTFWHTIASNFADN DNVIFDTNNEYHDMDESLVVQLNQAAIDGIRAAGATSQYIFVEGNSWTGAWTWTQVNDAMANLTDPQ NKIVYEMHQYLDSDGSGTSDQCVNSTIGQDRVESATAWLKQNGKKAILGEYAGGANSVCETAVTGML DYLANNTDVWTGAIWWAAGPWWGDYIFSMEPPSGIAYEQVLPLLQPYL BsuGlu103FULL, Bacillus subtilis (SEQ ID NO: 9) DDYSVVEEHGQLSISNGELVNERGEQVQLKGMSSHGLQWYGQFVNYESMKWLRDDWGITVFRAAMYT SSGGYIDDPSVKEKVKETVEAAIDLGIYVIIDWHILSDNDPNIYKEEAKDFFDEMSELYGDYPNVIY EIANEPNGSDVTWDNQIKPYAEEVIPVIRDNDPNNIVIVGTGTWSQDVHHAADNQLADPNVMYAFHF YAGTHGQNLRDQVDYALDQGAAIFVSEWGTSAATGDGGVFLDEAQVWIDFMDERNLSWANWSLTHKD ESSAALMPGANPTGGWTEAELSPSGTFVREKIRESASIPPSDPTPPSDPGEPDPGEPDPTPPSDPGE YPAWDSNQIYTNEIVYHNGQLWQAKWWTQNQEPGDPYGPWEPLKSDPDSGEPDPTPPSDPGEYPAWD SNQIYTNEIVYHNGQLWQAKWWTQNQEPGDPYGPWEPLN TreGlu2, Trichoderma reesei (SEQ ID NO: 10) QQTVWGQCGGIGWSGPTNCAPGSACSTLNPYYAQCIPGATTITTSTRPPSGPTTTTRATSTSSSTPP TSSGVRFAGVNIAGFDFGCTTDGTCVTSKVYPPLKNFTGSNNYPDGIGQMQHFVNDDGMTIFRLPVG WQYLVNNNLGGNLDSTSISKYDQLVQGCLSLGAYCIVDIHNYARWNGGIIGQGGPTNAQFTSLWSQL ASKYASQSRVWFGIMNEPHDVNINTWAATVQEVVTAIRNAGATSQFISLPGNDWQSAGAFISDGSAA ALSQVTNPDGSTTNLIFDVHKYLDSDNSGTHAECTTAINIDGAFSPLATWLRQNNRQAILTETGGGN VQSCIQDMCQQIQYLNQNSDVYLGYVGWGAGSFDSTYVLTETPTGSGNSWTDTSLVSSCLARK TreGlu3, Trichoderma reesei (SEQ ID NO: 11) QTSCDQWATFTGNGYTVSNNLWGASAGSGFGCVTAVSLSGGASWHADWQWSGGQNNVKSYQNSQI AIPQKRTVNSISSMPTTASWSYSGSNIRANVAYDLFTAANPNHVTYSGDYELMIWLGKYGDIGPI GSSQGTVNVGGQSWILYYGYNGAMQVYSFVAQINTTNYSGDVKNFFNYLRDNKGYNAAGQYVLSY QFGTEPFTGSGTLNVASWTASIN TreGlu4, Trichoderma reesei (SEQ ID NO: 12) HGHINDIVINGVWYQAYDPTTFPYESNPPIVVGWTAADLDNGFVSPDAYQNPDIICHKNATNAKGHA SVKAGDTILFQWVPVPWPHPGPIVDYLANCNGDCETVDKTTLEFFKIDGVGLLSGGDPGTWASDVLI SNNNTWVVKIPDNLAPGNYVLRHEIIALHSAGQANGAQNYPQCFNIAVSGSGSLQPSGVLGTDLYHA TDPGVLINIYTSPLNYIIPGPTVVSGLPTSVAQGSSAATATASATVPGGGSGPTSRTFTTARTTQAS SRPSSTPPATTSAPAGGPTQTLYGQCGGSGYSGPTRCAPPATCSTNPYYAQCLN TreGlu6, Trichoderma reesei (SEQ ID NO: 13) AFSWKNVKLGGGGGFVPGIIFHPKTKGVAYARTDIGGLYRLNADDSWTAVTDGIADNAGWHNWGIDAV ALDPQDDQKVYAAVGMYTNSWDPSNGAIIRSSDRGATWSFTNLPFKVGGNMPGRGAGERLAVDPANSN HYFGARSGNGLWKSTDGGVTFSKVSSFTATGTYIPDPSDSNGYNSDKQGLMWVTFDSTSSTTGGATSR IFVGTADNITASVYVSTNAGSTWSAVPGQPGKYFPHKAKLQPAEKALYLTYSWWPDAQLFRSTDSGTF WSPIWAWASYPTETYYYSISTPKAPWIKNNFIDVTSESPSDGLIKRLGWMIESLEIDPTDSNHWLYGT GMTIFGGHDLTNWDTRHNVSIQSLADGIEEFSVQDLASAPGGSELLAAVGDDNGFTFASRNDLGTSPQ TVWATPTWATSTSVDYAGNSVKSVVRVGNTAGTQQVAISSDGGATWSIDYAADTSMNGGTVAYSADGD TILWSTASSGVQRSQFQGSFASVSSLPAGAVIASDKKTNSVFYAGSGSTFYVSKDTGSSFTRGPKLGS AGTIRDIAAHPTTAGTLYVSTDVGIFRSTDSGTTFGQVSTALTNTYQIALGVGSGSNWNLYAFGTGPS GARLYASGDSGASWTDIQGSQGFGSIDSTKVAGSGSTAGQVYVGTNGRGVFYAQGTVGGGTGGTSSST KQSSSSTSSASSSTFLRSSVVSTTRASTVTSSRTSSAAGPTGSGVAGHYAQCGGIGWTGPTQCVAPYV CQKQNDYYYQCV TreGlu7, Trichoderma reesei (SEQ ID NO: 14) HGQVQNFTINGQYNQGFILDYYYQKQNTGHFPNVAGWYAEDLDLGFISPDQYTTPDIVCHKNAAPGAI SATAAAGSNIVFQWGPGVWPHPYGPIVTYVVECSGSCTTVNKNNLRWVKIQEAGINYNTQVWAQQDLI NQGNKWTVKIPSSLRPGNYVFRHELLAAHGASSANGMQNYPQCVNIAVTGSGTKALPAGTPATQLYKP TDPGILFNPYTTITSYTIPGPALWQG TreGlu8, Trichoderma reesei (SEQ ID NO: 15) GKIKYLGVAIPGIDFGCDIDGSCPTDTSSVPLLSYKGGDGAGQMKHFAEDDGLNVFRISATWQF VLNNTVDGKLDELNWGSYNKVVNACLETGAYCMIDMHNFARYNGGIIGQGGVSDDIFVDLWVQI AKYYEDNDKIIFGLMNEPHDLDIEIWAQTCQKVVTAIRKAGATSQMILLPGTNFASVETYVSTG SAEALGKITNPDGSTDLLYFDVHKYLDINNSGSHAECTFDNVDAFNDFADWLRQNKRQAIISET GASMEPSCMTAFCAQNKAISENSDVYIGFVGWGAGSFDTSYILTLTPLGKPGNYTDNKLMNECI LDQFTLDEKYRPTPTSISTAAEETATATATSDGDAPSTTKPIFREETASPTPNAVTKPSPDTSD SSDDDKDSAASMSAQGLTGTVLFTVAALGYMLVAF BsuGluC CBD, Bacillus subtilis (SEQ ID NO: 16) MKRSISIFITCLLITLLTMGGMIASPASAAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLKGISS HGLQWYGEYVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVII DWHILNDGNPNQNKEKAKEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIR KNDPDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVT EWGTSDASGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGASKTGGWRLSDLSA SGTFVRENILGTKDSTKDIPETPSKDKPTQENGISVQYRAGDGSMNSNQIRPQLQIKNNGNTTV DLKDVTARYWYKAKNKGQNFDCDYAQIGCGNVTHKFVTLHKPKQGADTYLELGFKNGTLAPGAS TGNIQLRLHNDDWSNYAQSGDYSFFKSNTFKTFKKITLYDQGKLIWGTEPN BsuXyn3, Bacillus subtilis xylanase variant (SEQ ID NO: 17) ASTDYWQNWTFGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYL TLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDDTTFTQYWSVR QSKRPTGSNATITFSNHVNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW BsuXyn4, Bacillus subtilis xylanase variant (SEQ ID NO: 18) ASTDYWQNWTDGYGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYL TLYGWTRSPLIEYYVVDSWGTYRPTGTYKGTVYSDGGWYDIYTATRDNAPSIDGDFTTFTQYWSVR QSKRPTGSNATITFSNHVNAWRSHGMDLGSNWAYQVMATEGYLSSGSSNVTVW

Sequence CWU 1

1

181302PRTAspergillus tubigensis 1Gln Ala Ser Val Ser Ile Asp Thr Lys Phe Lys Ala His Gly Lys Lys 1 5 10 15 Tyr Leu Gly Asn Ile Gly Asp Gln Tyr Thr Leu Thr Lys Asn Ser Lys 20 25 30 Thr Pro Ala Ile Ile Lys Ala Asp Phe Gly Ala Leu Thr Pro Glu Asn 35 40 45 Ser Met Lys Trp Asp Ala Thr Glu Pro Ser Arg Gly Gln Phe Ser Phe 50 55 60 Ser Gly Ser Asp Tyr Leu Val Asn Phe Ala Gln Ser Asn Asn Lys Leu 65 70 75 80 Ile Arg Gly His Thr Leu Val Trp His Ser Gln Leu Pro Ser Trp Val 85 90 95 Gln Ala Ile Thr Asp Lys Asn Thr Leu Ile Glu Val Met Lys Asn His 100 105 110 Ile Thr Thr Val Met Gln His Tyr Lys Gly Lys Ile Tyr Ala Trp Asp 115 120 125 Val Val Asn Glu Ile Phe Asn Glu Asp Gly Ser Leu Arg Asp Ser Val 130 135 140 Phe Tyr Gln Val Ile Gly Glu Asp Tyr Val Arg Ile Ala Phe Glu Thr 145 150 155 160 Ala Arg Ala Ala Asp Pro Asn Ala Lys Leu Tyr Ile Asn Asp Tyr Asn 165 170 175 Leu Asp Ser Ala Ser Tyr Pro Lys Leu Thr Gly Met Val Ser His Val 180 185 190 Lys Lys Trp Ile Glu Ala Gly Ile Pro Ile Asp Gly Ile Gly Ser Gln 195 200 205 Thr His Leu Ser Ala Gly Gly Gly Ala Gly Ile Ser Gly Ala Leu Asn 210 215 220 Ala Leu Ala Gly Ala Gly Thr Lys Glu Ile Ala Val Thr Glu Leu Asp 225 230 235 240 Ile Ala Gly Ala Ser Ser Thr Asp Tyr Val Glu Val Val Glu Ala Cys 245 250 255 Leu Asp Gln Pro Lys Cys Ile Gly Ile Thr Val Trp Gly Val Ala Asp 260 265 270 Pro Asp Ser Trp Arg Ser Ser Ser Thr Pro Leu Leu Phe Asp Ser Asn 275 280 285 Tyr Asn Pro Lys Pro Ala Tyr Thr Ala Ile Ala Asn Ala Leu 290 295 300 2386PRTGeosmithia emersonii 2Ala Gly Leu Asn Thr Ala Ala Lys Ala Ile Gly Leu Lys Tyr Phe Gly 1 5 10 15 Thr Ala Thr Asp Asn Pro Glu Leu Ser Asp Thr Ala Tyr Glu Thr Gln 20 25 30 Leu Asn Asn Thr Gln Asp Phe Gly Gln Leu Thr Pro Ala Asn Ser Met 35 40 45 Lys Trp Asp Ala Thr Glu Pro Glu Gln Asn Val Phe Thr Phe Ser Ala 50 55 60 Gly Asp Gln Ile Ala Asn Leu Ala Lys Ala Asn Gly Gln Met Leu Arg 65 70 75 80 Cys His Asn Leu Val Trp Tyr Asn Gln Leu Pro Ser Trp Val Thr Ser 85 90 95 Gly Ser Trp Thr Asn Glu Thr Leu Leu Ala Ala Met Lys Asn His Ile 100 105 110 Thr Asn Val Val Thr His Tyr Lys Gly Gln Cys Tyr Ala Trp Asp Val 115 120 125 Val Asn Glu Ala Leu Asn Asp Asp Gly Thr Tyr Arg Ser Asn Val Phe 130 135 140 Tyr Gln Tyr Ile Gly Glu Ala Tyr Ile Pro Ile Ala Phe Ala Thr Ala 145 150 155 160 Ala Ala Ala Asp Pro Asn Ala Lys Leu Tyr Tyr Asn Asp Tyr Asn Ile 165 170 175 Glu Tyr Pro Gly Ala Lys Ala Thr Ala Ala Gln Asn Leu Val Lys Leu 180 185 190 Val Gln Ser Tyr Gly Ala Arg Ile Asp Gly Val Gly Leu Gln Ser His 195 200 205 Phe Ile Val Gly Glu Thr Pro Ser Thr Ser Ser Gln Gln Gln Asn Met 210 215 220 Ala Ala Phe Thr Ala Leu Gly Val Glu Val Ala Ile Thr Glu Leu Asp 225 230 235 240 Ile Arg Met Gln Leu Pro Glu Thr Glu Ala Leu Leu Thr Gln Gln Ala 245 250 255 Thr Asp Tyr Gln Ser Thr Val Gln Ala Cys Ala Asn Thr Lys Gly Cys 260 265 270 Val Gly Ile Thr Val Trp Asp Trp Thr Asp Lys Tyr Ser Trp Val Pro 275 280 285 Ser Thr Phe Ser Gly Tyr Gly Asp Ala Cys Pro Trp Asp Ala Asn Tyr 290 295 300 Gln Lys Lys Pro Ala Tyr Glu Gly Ile Leu Thr Gly Leu Gly Gln Thr 305 310 315 320 Val Thr Ser Thr Thr Tyr Ile Ile Ser Pro Thr Thr Ser Val Gly Thr 325 330 335 Gly Thr Thr Thr Ser Ser Gly Gly Ser Gly Gly Thr Thr Gly Val Ala 340 345 350 Gln His Trp Glu Gln Cys Gly Gly Leu Gly Trp Thr Gly Pro Thr Val 355 360 365 Cys Ala Ser Gly Tyr Thr Cys Thr Val Ile Asn Glu Tyr Tyr Ser Gln 370 375 380 Cys Leu 385 3308PRTAspergillus tubigensis 3Glu Pro Ile Glu Pro Arg Gln Ala Ser Val Ser Ile Asp Thr Lys Phe 1 5 10 15 Lys Ala His Gly Lys Lys Tyr Leu Gly Asn Ile Gly Asp Gln Tyr Thr 20 25 30 Leu Thr Lys Asn Ser Lys Thr Pro Ala Ile Ile Lys Ala Asp Phe Gly 35 40 45 Ala Leu Thr Pro Glu Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Ser 50 55 60 Arg Gly Gln Phe Ser Phe Ser Gly Ser Asp Tyr Leu Val Asn Phe Ala 65 70 75 80 Gln Ser Asn Asn Lys Leu Ile Arg Gly His Thr Leu Val Trp His Ser 85 90 95 Gln Leu Pro Ser Trp Val Gln Ser Ile Thr Asp Lys Asn Thr Leu Ile 100 105 110 Glu Val Met Lys Asn His Ile Thr Thr Val Met Gln His Tyr Lys Gly 115 120 125 Lys Ile Tyr Ala Trp Asp Val Val Asn Glu Ile Phe Asn Glu Asp Gly 130 135 140 Ser Leu Arg Asp Ser Val Phe Tyr Lys Val Ile Gly Glu Asp Tyr Val 145 150 155 160 Arg Ile Ala Phe Glu Thr Ala Arg Ala Ala Asp Pro Asn Ala Lys Leu 165 170 175 Tyr Ile Asn Asp Tyr Asn Leu Asp Ser Ala Ser Tyr Pro Lys Leu Thr 180 185 190 Gly Met Val Ser His Val Lys Lys Trp Ile Ala Ala Gly Ile Pro Ile 195 200 205 Asp Gly Ile Gly Ser Gln Thr His Leu Ser Ala Gly Gly Gly Ala Gly 210 215 220 Ile Ser Gly Ala Leu Asn Ala Leu Ala Gly Ala Gly Thr Lys Glu Ile 225 230 235 240 Ala Val Thr Glu Leu Asp Ile Ala Gly Ala Ser Ser Thr Asp Tyr Val 245 250 255 Glu Val Val Glu Ala Cys Leu Asn Gln Pro Lys Cys Ile Gly Ile Thr 260 265 270 Val Trp Gly Val Ala Asp Pro Asp Ser Trp Arg Ser Ser Ser Thr Pro 275 280 285 Leu Leu Phe Asp Ser Asn Tyr Asn Pro Lys Pro Ala Tyr Thr Ala Ile 290 295 300 Ala Asn Ala Leu 305 4406PRTAspergillus aculeatus 4Met Val Gly Leu Leu Ser Ile Thr Ala Ala Leu Ala Ala Thr Val Leu 1 5 10 15 Pro Asn Ile Val Ser Ala Val Gly Leu Asp Gln Ala Ala Val Ala Lys 20 25 30 Gly Leu Gln Tyr Phe Gly Thr Ala Thr Asp Asn Pro Glu Leu Thr Asp 35 40 45 Ile Pro Tyr Val Thr Gln Leu Asn Asn Thr Ala Asp Phe Gly Gln Ile 50 55 60 Thr Pro Gly Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Ser Gln Gly 65 70 75 80 Thr Phe Thr Phe Thr Lys Gly Asp Val Ile Ala Asp Leu Ala Glu Gly 85 90 95 Asn Gly Gln Tyr Leu Arg Cys His Thr Leu Val Trp Tyr Asn Gln Leu 100 105 110 Pro Ser Trp Val Thr Ser Gly Thr Trp Thr Asn Ala Thr Leu Thr Ala 115 120 125 Ala Leu Lys Asn His Ile Thr Asn Val Val Ser His Tyr Lys Gly Lys 130 135 140 Cys Leu His Trp Asp Val Val Asn Glu Ala Leu Asn Asp Asp Gly Thr 145 150 155 160 Tyr Arg Thr Asn Ile Phe Tyr Thr Thr Ile Gly Glu Ala Tyr Ile Pro 165 170 175 Ile Ala Phe Ala Ala Ala Ala Ala Ala Asp Pro Asp Ala Lys Leu Phe 180 185 190 Tyr Asn Asp Tyr Asn Leu Glu Tyr Gly Gly Ala Lys Ala Ala Ser Ala 195 200 205 Arg Ala Ile Val Gln Leu Val Lys Asn Ala Gly Ala Lys Ile Asp Gly 210 215 220 Val Gly Leu Gln Ala His Phe Ser Val Gly Thr Val Pro Ser Thr Ser 225 230 235 240 Ser Leu Val Ser Val Leu Gln Ser Phe Thr Ala Leu Gly Val Glu Val 245 250 255 Ala Tyr Thr Glu Ala Asp Val Arg Ile Leu Leu Pro Thr Thr Ala Thr 260 265 270 Thr Leu Ala Gln Gln Ser Ser Asp Phe Gln Ala Leu Val Gln Ser Cys 275 280 285 Val Gln Thr Thr Gly Cys Val Gly Phe Thr Ile Trp Asp Trp Thr Asp 290 295 300 Lys Tyr Ser Trp Val Pro Ser Thr Phe Ser Gly Tyr Gly Ala Ala Leu 305 310 315 320 Pro Trp Asp Glu Asn Leu Val Lys Lys Pro Ala Tyr Asn Gly Leu Leu 325 330 335 Ala Gly Met Gly Val Thr Val Thr Thr Thr Thr Thr Thr Thr Thr Ala 340 345 350 Thr Ala Thr Gly Lys Thr Thr Thr Thr Thr Thr Gly Ala Thr Ser Thr 355 360 365 Gly Thr Thr Ala Ala His Trp Gly Gln Cys Gly Gly Leu Asn Trp Ser 370 375 380 Gly Pro Thr Ala Cys Ala Thr Gly Tyr Thr Cys Thr Tyr Val Asn Asp 385 390 395 400 Tyr Tyr Ser Gln Cys Leu 405 5347PRTTrichoderma reesei 5Met Lys Ala Asn Val Ile Leu Cys Leu Leu Ala Pro Leu Val Ala Ala 1 5 10 15 Leu Pro Thr Glu Thr Ile His Leu Asp Pro Glu Leu Ala Ala Leu Arg 20 25 30 Ala Asn Leu Thr Glu Arg Thr Ala Asp Leu Trp Asp Arg Gln Ala Ser 35 40 45 Gln Ser Ile Asp Gln Leu Ile Lys Arg Lys Gly Lys Leu Tyr Phe Gly 50 55 60 Thr Ala Thr Asp Arg Gly Leu Leu Gln Arg Glu Lys Asn Ala Ala Ile 65 70 75 80 Ile Gln Ala Asp Leu Gly Gln Val Thr Pro Glu Asn Ser Met Lys Trp 85 90 95 Gln Ser Leu Glu Asn Asn Gln Gly Gln Leu Asn Trp Gly Asp Ala Asp 100 105 110 Tyr Leu Val Asn Phe Ala Gln Gln Asn Gly Lys Ser Ile Arg Gly His 115 120 125 Thr Leu Ile Trp His Ser Gln Leu Pro Ala Trp Val Asn Asn Ile Asn 130 135 140 Asn Ala Asp Thr Leu Arg Gln Val Ile Arg Thr His Val Ser Thr Val 145 150 155 160 Val Gly Arg Tyr Lys Gly Lys Ile Arg Ala Trp Asp Val Val Asn Glu 165 170 175 Ile Phe Asn Glu Asp Gly Thr Leu Arg Ser Ser Val Phe Ser Arg Leu 180 185 190 Leu Gly Glu Glu Phe Val Ser Ile Ala Phe Arg Ala Ala Arg Asp Ala 195 200 205 Asp Pro Ser Ala Arg Leu Tyr Ile Asn Asp Tyr Asn Leu Asp Arg Ala 210 215 220 Asn Tyr Gly Lys Val Asn Gly Leu Lys Thr Tyr Val Ser Lys Trp Ile 225 230 235 240 Ser Gln Gly Val Pro Ile Asp Gly Ile Gly Ser Gln Ser His Leu Ser 245 250 255 Gly Gly Gly Gly Ser Gly Thr Leu Gly Ala Leu Gln Gln Leu Ala Thr 260 265 270 Val Pro Val Thr Glu Leu Ala Ile Thr Glu Leu Asp Ile Gln Gly Ala 275 280 285 Pro Thr Thr Asp Tyr Thr Gln Val Val Gln Ala Cys Leu Ser Val Ser 290 295 300 Lys Cys Val Gly Ile Thr Val Trp Gly Ile Ser Asp Lys Asp Ser Trp 305 310 315 320 Arg Ala Ser Thr Asn Pro Leu Leu Phe Asp Ala Asn Phe Asn Pro Lys 325 330 335 Pro Ala Tyr Asn Ser Ile Val Gly Ile Leu Gln 340 345 6191PRTTrichoderma reesei 6Gln Cys Ile Gln Pro Gly Thr Gly Tyr Asn Asn Gly Tyr Phe Tyr Ser 1 5 10 15 Tyr Trp Asn Asp Gly His Gly Gly Val Thr Tyr Cys Asn Gly Pro Gly 20 25 30 Gly Gln Phe Ser Val Asn Trp Ser Asn Ser Gly Asn Phe Val Gly Gly 35 40 45 Lys Gly Trp Gln Pro Gly Thr Lys Asn Arg Val Ile Asn Phe Ser Gly 50 55 60 Ser Tyr Asn Pro Asn Gly Asn Ser Tyr Leu Ser Val Tyr Gly Trp Ser 65 70 75 80 Arg Asn Pro Leu Ile Glu Tyr Tyr Ile Val Glu Asn Phe Gly Thr Tyr 85 90 95 Asn Pro Ser Thr Gly Ala Thr Lys Leu Gly Glu Val Thr Ser Asp Gly 100 105 110 Ser Val Tyr Asp Ile Tyr Arg Thr Gln Arg Val Asn Gln Pro Ser Ile 115 120 125 Ile Gly Thr Ala Thr Phe Tyr Gln Tyr Trp Ser Val Arg Arg Asn His 130 135 140 Arg Ser Ser Gly Ser Val Asn Thr Ala Asn His Phe Asn Ala Trp Ala 145 150 155 160 Gln Gln Gly Leu Thr Leu Gly Thr Met Asp Tyr Gln Ile Val Ala Val 165 170 175 Glu Gly Tyr Phe Ser Ser Gly Ser Ala Ser Ile Thr Val Ser Asp 180 185 190 7214PRTBacillus subtilis 7Gln Thr Gly Gly Ser Phe Phe Asp Pro Phe Asn Gly Tyr Asn Ser Gly 1 5 10 15 Phe Trp Gln Lys Ala Asp Gly Tyr Ser Asn Gly Asn Met Phe Asn Cys 20 25 30 Thr Trp Arg Ala Asn Asn Val Ser Met Thr Ser Leu Gly Glu Met Arg 35 40 45 Leu Ala Leu Thr Ser Pro Ala Tyr Asn Lys Phe Asp Cys Gly Glu Asn 50 55 60 Arg Ser Val Gln Thr Tyr Gly Tyr Gly Leu Tyr Glu Val Arg Met Lys 65 70 75 80 Pro Ala Lys Asn Thr Gly Ile Val Ser Ser Phe Phe Thr Tyr Thr Gly 85 90 95 Pro Thr Asp Gly Thr Pro Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly 100 105 110 Lys Asp Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Ala Gly 115 120 125 Asn His Glu Lys Ile Val Asp Leu Gly Phe Asp Ala Ala Asn Ala Tyr 130 135 140 His Thr Tyr Ala Phe Asp Trp Gln Pro Asn Ser Ile Lys Trp Tyr Val 145 150 155 160 Asp Gly Gln Leu Lys His Thr Ala Thr Asn Gln Ile Pro Thr Thr Pro 165 170 175 Gly Lys Ile Met Met Asn Leu Trp Asn Gly Thr Gly Val Asp Glu Trp 180 185 190 Leu Gly Ser Tyr Asn Gly Val Asn Pro Leu Tyr Ala His Tyr Asp Trp 195 200 205 Val Arg Tyr Thr Lys Lys 210 8316PRTGeosmithia emersonii 8Ala Pro Val Lys Glu Lys Gly Ile Lys Lys Arg Ala Ser Pro Phe Gln 1 5 10 15 Trp Phe Gly Ser Asn Glu Ser Gly Ala Glu Phe Gly Asn Asn Asn Ile 20 25 30 Pro Gly Val Glu Gly Thr Asp Tyr Thr Phe Pro Asn Thr Ser Ala Ile 35 40 45 Gln Ile Leu Ile Asp Gln Gly Met Asn Ile Phe Arg Val Pro Phe Leu 50 55 60 Met Glu Arg Met Val Pro Asn Gln Met Thr Gly Pro Val Asp Ser Ala 65 70 75 80 Tyr Phe Gln Gly Tyr Ser Gln Val Ile Asn Tyr Ile Thr Ser His Gly 85 90 95 Ala Ser Ala Val Ile Asp Pro His Asn Phe Gly Arg Tyr Tyr Asn Asn 100 105 110 Ile Ile Ser Ser Pro Ser Asp Phe Gln Thr Phe Trp His Thr Ile

Ala 115 120 125 Ser Asn Phe Ala Asp Asn Asp Asn Val Ile Phe Asp Thr Asn Asn Glu 130 135 140 Tyr His Asp Met Asp Glu Ser Leu Val Val Gln Leu Asn Gln Ala Ala 145 150 155 160 Ile Asp Gly Ile Arg Ala Ala Gly Ala Thr Ser Gln Tyr Ile Phe Val 165 170 175 Glu Gly Asn Ser Trp Thr Gly Ala Trp Thr Trp Thr Gln Val Asn Asp 180 185 190 Ala Met Ala Asn Leu Thr Asp Pro Gln Asn Lys Ile Val Tyr Glu Met 195 200 205 His Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Asp Gln Cys Val 210 215 220 Asn Ser Thr Ile Gly Gln Asp Arg Val Glu Ser Ala Thr Ala Trp Leu 225 230 235 240 Lys Gln Asn Gly Lys Lys Ala Ile Leu Gly Glu Tyr Ala Gly Gly Ala 245 250 255 Asn Ser Val Cys Glu Thr Ala Val Thr Gly Met Leu Asp Tyr Leu Ala 260 265 270 Asn Asn Thr Asp Val Trp Thr Gly Ala Ile Trp Trp Ala Ala Gly Pro 275 280 285 Trp Trp Gly Asp Tyr Ile Phe Ser Met Glu Pro Pro Ser Gly Ile Ala 290 295 300 Tyr Glu Gln Val Leu Pro Leu Leu Gln Pro Tyr Leu 305 310 315 9441PRTBacillus subtilis 9Asp Asp Tyr Ser Val Val Glu Glu His Gly Gln Leu Ser Ile Ser Asn 1 5 10 15 Gly Glu Leu Val Asn Glu Arg Gly Glu Gln Val Gln Leu Lys Gly Met 20 25 30 Ser Ser His Gly Leu Gln Trp Tyr Gly Gln Phe Val Asn Tyr Glu Ser 35 40 45 Met Lys Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala 50 55 60 Met Tyr Thr Ser Ser Gly Gly Tyr Ile Asp Asp Pro Ser Val Lys Glu 65 70 75 80 Lys Val Lys Glu Thr Val Glu Ala Ala Ile Asp Leu Gly Ile Tyr Val 85 90 95 Ile Ile Asp Trp His Ile Leu Ser Asp Asn Asp Pro Asn Ile Tyr Lys 100 105 110 Glu Glu Ala Lys Asp Phe Phe Asp Glu Met Ser Glu Leu Tyr Gly Asp 115 120 125 Tyr Pro Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Ser Asp 130 135 140 Val Thr Trp Asp Asn Gln Ile Lys Pro Tyr Ala Glu Glu Val Ile Pro 145 150 155 160 Val Ile Arg Asp Asn Asp Pro Asn Asn Ile Val Ile Val Gly Thr Gly 165 170 175 Thr Trp Ser Gln Asp Val His His Ala Ala Asp Asn Gln Leu Ala Asp 180 185 190 Pro Asn Val Met Tyr Ala Phe His Phe Tyr Ala Gly Thr His Gly Gln 195 200 205 Asn Leu Arg Asp Gln Val Asp Tyr Ala Leu Asp Gln Gly Ala Ala Ile 210 215 220 Phe Val Ser Glu Trp Gly Thr Ser Ala Ala Thr Gly Asp Gly Gly Val 225 230 235 240 Phe Leu Asp Glu Ala Gln Val Trp Ile Asp Phe Met Asp Glu Arg Asn 245 250 255 Leu Ser Trp Ala Asn Trp Ser Leu Thr His Lys Asp Glu Ser Ser Ala 260 265 270 Ala Leu Met Pro Gly Ala Asn Pro Thr Gly Gly Trp Thr Glu Ala Glu 275 280 285 Leu Ser Pro Ser Gly Thr Phe Val Arg Glu Lys Ile Arg Glu Ser Ala 290 295 300 Ser Ile Pro Pro Ser Asp Pro Thr Pro Pro Ser Asp Pro Gly Glu Pro 305 310 315 320 Asp Pro Gly Glu Pro Asp Pro Thr Pro Pro Ser Asp Pro Gly Glu Tyr 325 330 335 Pro Ala Trp Asp Ser Asn Gln Ile Tyr Thr Asn Glu Ile Val Tyr His 340 345 350 Asn Gly Gln Leu Trp Gln Ala Lys Trp Trp Thr Gln Asn Gln Glu Pro 355 360 365 Gly Asp Pro Tyr Gly Pro Trp Glu Pro Leu Lys Ser Asp Pro Asp Ser 370 375 380 Gly Glu Pro Asp Pro Thr Pro Pro Ser Asp Pro Gly Glu Tyr Pro Ala 385 390 395 400 Trp Asp Ser Asn Gln Ile Tyr Thr Asn Glu Ile Val Tyr His Asn Gly 405 410 415 Gln Leu Trp Gln Ala Lys Trp Trp Thr Gln Asn Gln Glu Pro Gly Asp 420 425 430 Pro Tyr Gly Pro Trp Glu Pro Leu Asn 435 440 10397PRTTrichoderma reesei 10Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Ile Gly Trp Ser Gly Pro 1 5 10 15 Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Thr Leu Asn Pro Tyr Tyr 20 25 30 Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr Thr Ser Thr Arg Pro 35 40 45 Pro Ser Gly Pro Thr Thr Thr Thr Arg Ala Thr Ser Thr Ser Ser Ser 50 55 60 Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala Gly Val Asn Ile Ala 65 70 75 80 Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr Cys Val Thr Ser Lys 85 90 95 Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser Asn Asn Tyr Pro Asp 100 105 110 Gly Ile Gly Gln Met Gln His Phe Val Asn Asp Asp Gly Met Thr Ile 115 120 125 Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val Asn Asn Asn Leu Gly 130 135 140 Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr Asp Gln Leu Val Gln 145 150 155 160 Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile Val Asp Ile His Asn Tyr 165 170 175 Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly Gly Pro Thr Asn Ala 180 185 190 Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser Lys Tyr Ala Ser Gln 195 200 205 Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro His Asp Val Asn Ile 210 215 220 Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val Thr Ala Ile Arg Asn 225 230 235 240 Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro Gly Asn Asp Trp Gln 245 250 255 Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala Ala Ala Leu Ser Gln 260 265 270 Val Thr Asn Pro Asp Gly Ser Thr Thr Asn Leu Ile Phe Asp Val His 275 280 285 Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His Ala Glu Cys Thr Thr 290 295 300 Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu Ala Thr Trp Leu Arg Gln 305 310 315 320 Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly Gly Gly Asn Val Gln 325 330 335 Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln Tyr Leu Asn Gln Asn 340 345 350 Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly Ala Gly Ser Phe Asp 355 360 365 Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Gly Ser Gly Asn Ser Trp 370 375 380 Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ala Arg Lys 385 390 395 11218PRTTrichoderma reesei 11Gln Thr Ser Cys Asp Gln Trp Ala Thr Phe Thr Gly Asn Gly Tyr Thr 1 5 10 15 Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gly Ser Gly Phe Gly Cys 20 25 30 Val Thr Ala Val Ser Leu Ser Gly Gly Ala Ser Trp His Ala Asp Trp 35 40 45 Gln Trp Ser Gly Gly Gln Asn Asn Val Lys Ser Tyr Gln Asn Ser Gln 50 55 60 Ile Ala Ile Pro Gln Lys Arg Thr Val Asn Ser Ile Ser Ser Met Pro 65 70 75 80 Thr Thr Ala Ser Trp Ser Tyr Ser Gly Ser Asn Ile Arg Ala Asn Val 85 90 95 Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro Asn His Val Thr Tyr Ser 100 105 110 Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp Ile Gly 115 120 125 Pro Ile Gly Ser Ser Gln Gly Thr Val Asn Val Gly Gly Gln Ser Trp 130 135 140 Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gln Val Tyr Ser Phe Val 145 150 155 160 Ala Gln Thr Asn Thr Thr Asn Tyr Ser Gly Asp Val Lys Asn Phe Phe 165 170 175 Asn Tyr Leu Arg Asp Asn Lys Gly Tyr Asn Ala Ala Gly Gln Tyr Val 180 185 190 Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe Thr Gly Ser Gly Thr Leu 195 200 205 Asn Val Ala Ser Trp Thr Ala Ser Ile Asn 210 215 12322PRTTrichoderma reesei 12His Gly His Ile Asn Asp Ile Val Ile Asn Gly Val Trp Tyr Gln Ala 1 5 10 15 Tyr Asp Pro Thr Thr Phe Pro Tyr Glu Ser Asn Pro Pro Ile Val Val 20 25 30 Gly Trp Thr Ala Ala Asp Leu Asp Asn Gly Phe Val Ser Pro Asp Ala 35 40 45 Tyr Gln Asn Pro Asp Ile Ile Cys His Lys Asn Ala Thr Asn Ala Lys 50 55 60 Gly His Ala Ser Val Lys Ala Gly Asp Thr Ile Leu Phe Gln Trp Val 65 70 75 80 Pro Val Pro Trp Pro His Pro Gly Pro Ile Val Asp Tyr Leu Ala Asn 85 90 95 Cys Asn Gly Asp Cys Glu Thr Val Asp Lys Thr Thr Leu Glu Phe Phe 100 105 110 Lys Ile Asp Gly Val Gly Leu Leu Ser Gly Gly Asp Pro Gly Thr Trp 115 120 125 Ala Ser Asp Val Leu Ile Ser Asn Asn Asn Thr Trp Val Val Lys Ile 130 135 140 Pro Asp Asn Leu Ala Pro Gly Asn Tyr Val Leu Arg His Glu Ile Ile 145 150 155 160 Ala Leu His Ser Ala Gly Gln Ala Asn Gly Ala Gln Asn Tyr Pro Gln 165 170 175 Cys Phe Asn Ile Ala Val Ser Gly Ser Gly Ser Leu Gln Pro Ser Gly 180 185 190 Val Leu Gly Thr Asp Leu Tyr His Ala Thr Asp Pro Gly Val Leu Ile 195 200 205 Asn Ile Tyr Thr Ser Pro Leu Asn Tyr Ile Ile Pro Gly Pro Thr Val 210 215 220 Val Ser Gly Leu Pro Thr Ser Val Ala Gln Gly Ser Ser Ala Ala Thr 225 230 235 240 Ala Thr Ala Ser Ala Thr Val Pro Gly Gly Gly Ser Gly Pro Thr Ser 245 250 255 Arg Thr Thr Thr Thr Ala Arg Thr Thr Gln Ala Ser Ser Arg Pro Ser 260 265 270 Ser Thr Pro Pro Ala Thr Thr Ser Ala Pro Ala Gly Gly Pro Thr Gln 275 280 285 Thr Leu Tyr Gly Gln Cys Gly Gly Ser Gly Tyr Ser Gly Pro Thr Arg 290 295 300 Cys Ala Pro Pro Ala Thr Cys Ser Thr Asn Pro Tyr Tyr Ala Gln Cys 305 310 315 320 Leu Asn 13761PRTTrichoderma reesei 13Ala Phe Ser Trp Lys Asn Val Lys Leu Gly Gly Gly Gly Gly Phe Val 1 5 10 15 Pro Gly Ile Ile Phe His Pro Lys Thr Lys Gly Val Ala Tyr Ala Arg 20 25 30 Thr Asp Ile Gly Gly Leu Tyr Arg Leu Asn Ala Asp Asp Ser Trp Thr 35 40 45 Ala Val Thr Asp Gly Ile Ala Asp Asn Ala Gly Trp His Asn Trp Gly 50 55 60 Ile Asp Ala Val Ala Leu Asp Pro Gln Asp Asp Gln Lys Val Tyr Ala 65 70 75 80 Ala Val Gly Met Tyr Thr Asn Ser Trp Asp Pro Ser Asn Gly Ala Ile 85 90 95 Ile Arg Ser Ser Asp Arg Gly Ala Thr Trp Ser Phe Thr Asn Leu Pro 100 105 110 Phe Lys Val Gly Gly Asn Met Pro Gly Arg Gly Ala Gly Glu Arg Leu 115 120 125 Ala Val Asp Pro Ala Asn Ser Asn Ile Ile Tyr Phe Gly Ala Arg Ser 130 135 140 Gly Asn Gly Leu Trp Lys Ser Thr Asp Gly Gly Val Thr Phe Ser Lys 145 150 155 160 Val Ser Ser Phe Thr Ala Thr Gly Thr Tyr Ile Pro Asp Pro Ser Asp 165 170 175 Ser Asn Gly Tyr Asn Ser Asp Lys Gln Gly Leu Met Trp Val Thr Phe 180 185 190 Asp Ser Thr Ser Ser Thr Thr Gly Gly Ala Thr Ser Arg Ile Phe Val 195 200 205 Gly Thr Ala Asp Asn Ile Thr Ala Ser Val Tyr Val Ser Thr Asn Ala 210 215 220 Gly Ser Thr Trp Ser Ala Val Pro Gly Gln Pro Gly Lys Tyr Phe Pro 225 230 235 240 His Lys Ala Lys Leu Gln Pro Ala Glu Lys Ala Leu Tyr Leu Thr Tyr 245 250 255 Ser Trp Trp Pro Asp Ala Gln Leu Phe Arg Ser Thr Asp Ser Gly Thr 260 265 270 Thr Trp Ser Pro Ile Trp Ala Trp Ala Ser Tyr Pro Thr Glu Thr Tyr 275 280 285 Tyr Tyr Ser Ile Ser Thr Pro Lys Ala Pro Trp Ile Lys Asn Asn Phe 290 295 300 Ile Asp Val Thr Ser Glu Ser Pro Ser Asp Gly Leu Ile Lys Arg Leu 305 310 315 320 Gly Trp Met Ile Glu Ser Leu Glu Ile Asp Pro Thr Asp Ser Asn His 325 330 335 Trp Leu Tyr Gly Thr Gly Met Thr Ile Phe Gly Gly His Asp Leu Thr 340 345 350 Asn Trp Asp Thr Arg His Asn Val Ser Ile Gln Ser Leu Ala Asp Gly 355 360 365 Ile Glu Glu Phe Ser Val Gln Asp Leu Ala Ser Ala Pro Gly Gly Ser 370 375 380 Glu Leu Leu Ala Ala Val Gly Asp Asp Asn Gly Phe Thr Phe Ala Ser 385 390 395 400 Arg Asn Asp Leu Gly Thr Ser Pro Gln Thr Val Trp Ala Thr Pro Thr 405 410 415 Trp Ala Thr Ser Thr Ser Val Asp Tyr Ala Gly Asn Ser Val Lys Ser 420 425 430 Val Val Arg Val Gly Asn Thr Ala Gly Thr Gln Gln Val Ala Ile Ser 435 440 445 Ser Asp Gly Gly Ala Thr Trp Ser Ile Asp Tyr Ala Ala Asp Thr Ser 450 455 460 Met Asn Gly Gly Thr Val Ala Tyr Ser Ala Asp Gly Asp Thr Ile Leu 465 470 475 480 Trp Ser Thr Ala Ser Ser Gly Val Gln Arg Ser Gln Phe Gln Gly Ser 485 490 495 Phe Ala Ser Val Ser Ser Leu Pro Ala Gly Ala Val Ile Ala Ser Asp 500 505 510 Lys Lys Thr Asn Ser Val Phe Tyr Ala Gly Ser Gly Ser Thr Phe Tyr 515 520 525 Val Ser Lys Asp Thr Gly Ser Ser Phe Thr Arg Gly Pro Lys Leu Gly 530 535 540 Ser Ala Gly Thr Ile Arg Asp Ile Ala Ala His Pro Thr Thr Ala Gly 545 550 555 560 Thr Leu Tyr Val Ser Thr Asp Val Gly Ile Phe Arg Ser Thr Asp Ser 565 570 575 Gly Thr Thr Phe Gly Gln Val Ser Thr Ala Leu Thr Asn Thr Tyr Gln 580 585 590 Ile Ala Leu Gly Val Gly Ser Gly Ser Asn Trp Asn Leu Tyr Ala Phe 595 600 605 Gly Thr Gly Pro Ser Gly Ala Arg Leu Tyr Ala Ser Gly Asp Ser Gly 610 615 620 Ala Ser Trp Thr Asp Ile Gln Gly Ser Gln Gly Phe Gly Ser Ile Asp 625 630 635 640 Ser Thr Lys Val Ala Gly Ser Gly Ser Thr Ala Gly Gln Val Tyr Val 645 650 655 Gly Thr Asn Gly Arg Gly Val Phe Tyr Ala Gln Gly Thr Val Gly Gly 660 665 670 Gly Thr Gly Gly Thr Ser Ser Ser Thr Lys Gln Ser Ser Ser Ser Thr 675 680 685 Ser Ser Ala Ser Ser Ser Thr Thr Leu Arg Ser Ser Val Val Ser Thr 690 695 700 Thr Arg Ala Ser Thr Val Thr Ser Ser Arg Thr Ser Ser Ala Ala Gly 705 710

715 720 Pro Thr Gly Ser Gly Val Ala Gly His Tyr Ala Gln Cys Gly Gly Ile 725 730 735 Gly Trp Thr Gly Pro Thr Gln Cys Val Ala Pro Tyr Val Cys Gln Lys 740 745 750 Gln Asn Asp Tyr Tyr Tyr Gln Cys Val 755 760 14230PRTTrichoderma reesei 14His Gly Gln Val Gln Asn Phe Thr Ile Asn Gly Gln Tyr Asn Gln Gly 1 5 10 15 Phe Ile Leu Asp Tyr Tyr Tyr Gln Lys Gln Asn Thr Gly His Phe Pro 20 25 30 Asn Val Ala Gly Trp Tyr Ala Glu Asp Leu Asp Leu Gly Phe Ile Ser 35 40 45 Pro Asp Gln Tyr Thr Thr Pro Asp Ile Val Cys His Lys Asn Ala Ala 50 55 60 Pro Gly Ala Ile Ser Ala Thr Ala Ala Ala Gly Ser Asn Ile Val Phe 65 70 75 80 Gln Trp Gly Pro Gly Val Trp Pro His Pro Tyr Gly Pro Ile Val Thr 85 90 95 Tyr Val Val Glu Cys Ser Gly Ser Cys Thr Thr Val Asn Lys Asn Asn 100 105 110 Leu Arg Trp Val Lys Ile Gln Glu Ala Gly Ile Asn Tyr Asn Thr Gln 115 120 125 Val Trp Ala Gln Gln Asp Leu Ile Asn Gln Gly Asn Lys Trp Thr Val 130 135 140 Lys Ile Pro Ser Ser Leu Arg Pro Gly Asn Tyr Val Phe Arg His Glu 145 150 155 160 Leu Leu Ala Ala His Gly Ala Ser Ser Ala Asn Gly Met Gln Asn Tyr 165 170 175 Pro Gln Cys Val Asn Ile Ala Val Thr Gly Ser Gly Thr Lys Ala Leu 180 185 190 Pro Ala Gly Thr Pro Ala Thr Gln Leu Tyr Lys Pro Thr Asp Pro Gly 195 200 205 Ile Leu Phe Asn Pro Tyr Thr Thr Ile Thr Ser Tyr Thr Ile Pro Gly 210 215 220 Pro Ala Leu Trp Gln Gly 225 230 15419PRTTrichoderma reesei 15Gly Lys Ile Lys Tyr Leu Gly Val Ala Ile Pro Gly Ile Asp Phe Gly 1 5 10 15 Cys Asp Ile Asp Gly Ser Cys Pro Thr Asp Thr Ser Ser Val Pro Leu 20 25 30 Leu Ser Tyr Lys Gly Gly Asp Gly Ala Gly Gln Met Lys His Phe Ala 35 40 45 Glu Asp Asp Gly Leu Asn Val Phe Arg Ile Ser Ala Thr Trp Gln Phe 50 55 60 Val Leu Asn Asn Thr Val Asp Gly Lys Leu Asp Glu Leu Asn Trp Gly 65 70 75 80 Ser Tyr Asn Lys Val Val Asn Ala Cys Leu Glu Thr Gly Ala Tyr Cys 85 90 95 Met Ile Asp Met His Asn Phe Ala Arg Tyr Asn Gly Gly Ile Ile Gly 100 105 110 Gln Gly Gly Val Ser Asp Asp Ile Phe Val Asp Leu Trp Val Gln Ile 115 120 125 Ala Lys Tyr Tyr Glu Asp Asn Asp Lys Ile Ile Phe Gly Leu Met Asn 130 135 140 Glu Pro His Asp Leu Asp Ile Glu Ile Trp Ala Gln Thr Cys Gln Lys 145 150 155 160 Val Val Thr Ala Ile Arg Lys Ala Gly Ala Thr Ser Gln Met Ile Leu 165 170 175 Leu Pro Gly Thr Asn Phe Ala Ser Val Glu Thr Tyr Val Ser Thr Gly 180 185 190 Ser Ala Glu Ala Leu Gly Lys Ile Thr Asn Pro Asp Gly Ser Thr Asp 195 200 205 Leu Leu Tyr Phe Asp Val His Lys Tyr Leu Asp Ile Asn Asn Ser Gly 210 215 220 Ser His Ala Glu Cys Thr Thr Asp Asn Val Asp Ala Phe Asn Asp Phe 225 230 235 240 Ala Asp Trp Leu Arg Gln Asn Lys Arg Gln Ala Ile Ile Ser Glu Thr 245 250 255 Gly Ala Ser Met Glu Pro Ser Cys Met Thr Ala Phe Cys Ala Gln Asn 260 265 270 Lys Ala Ile Ser Glu Asn Ser Asp Val Tyr Ile Gly Phe Val Gly Trp 275 280 285 Gly Ala Gly Ser Phe Asp Thr Ser Tyr Ile Leu Thr Leu Thr Pro Leu 290 295 300 Gly Lys Pro Gly Asn Tyr Thr Asp Asn Lys Leu Met Asn Glu Cys Ile 305 310 315 320 Leu Asp Gln Phe Thr Leu Asp Glu Lys Tyr Arg Pro Thr Pro Thr Ser 325 330 335 Ile Ser Thr Ala Ala Glu Glu Thr Ala Thr Ala Thr Ala Thr Ser Asp 340 345 350 Gly Asp Ala Pro Ser Thr Thr Lys Pro Ile Phe Arg Glu Glu Thr Ala 355 360 365 Ser Pro Thr Pro Asn Ala Val Thr Lys Pro Ser Pro Asp Thr Ser Asp 370 375 380 Ser Ser Asp Asp Asp Lys Asp Ser Ala Ala Ser Met Ser Ala Gln Gly 385 390 395 400 Leu Thr Gly Thr Val Leu Phe Thr Val Ala Ala Leu Gly Tyr Met Leu 405 410 415 Val Ala Phe 16499PRTBacillus subtilis 16Met Lys Arg Ser Ile Ser Ile Phe Ile Thr Cys Leu Leu Ile Thr Leu 1 5 10 15 Leu Thr Met Gly Gly Met Ile Ala Ser Pro Ala Ser Ala Ala Gly Thr 20 25 30 Lys Thr Pro Val Ala Lys Asn Gly Gln Leu Ser Ile Lys Gly Thr Gln 35 40 45 Leu Val Asn Arg Asp Gly Lys Ala Val Gln Leu Lys Gly Ile Ser Ser 50 55 60 His Gly Leu Gln Trp Tyr Gly Glu Tyr Val Asn Lys Asp Ser Leu Lys 65 70 75 80 Trp Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr 85 90 95 Thr Ala Asp Gly Gly Tyr Ile Asp Asn Pro Ser Val Lys Asn Lys Val 100 105 110 Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val Ile Ile 115 120 125 Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln Asn Lys Glu Lys 130 135 140 Ala Lys Glu Phe Phe Lys Glu Met Ser Ser Leu Tyr Gly Asn Thr Pro 145 150 155 160 Asn Val Ile Tyr Glu Ile Ala Asn Glu Pro Asn Gly Asp Val Asn Trp 165 170 175 Lys Arg Asp Ile Lys Pro Tyr Ala Glu Glu Val Ile Ser Val Ile Arg 180 185 190 Lys Asn Asp Pro Asp Asn Ile Ile Ile Val Gly Thr Gly Thr Trp Ser 195 200 205 Gln Asp Val Asn Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val 210 215 220 Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg 225 230 235 240 Asp Lys Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr 245 250 255 Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly Gly Val Phe Leu Asp 260 265 270 Gln Ser Arg Glu Trp Leu Lys Tyr Leu Asp Ser Lys Thr Ile Ser Trp 275 280 285 Val Asn Trp Asn Leu Ser Asp Lys Gln Glu Ser Ser Ser Ala Leu Lys 290 295 300 Pro Gly Ala Ser Lys Thr Gly Gly Trp Arg Leu Ser Asp Leu Ser Ala 305 310 315 320 Ser Gly Thr Phe Val Arg Glu Asn Ile Leu Gly Thr Lys Asp Ser Thr 325 330 335 Lys Asp Ile Pro Glu Thr Pro Ser Lys Asp Lys Pro Thr Gln Glu Asn 340 345 350 Gly Ile Ser Val Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn 355 360 365 Gln Ile Arg Pro Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Val 370 375 380 Asp Leu Lys Asp Val Thr Ala Arg Tyr Trp Tyr Lys Ala Lys Asn Lys 385 390 395 400 Gly Gln Asn Phe Asp Cys Asp Tyr Ala Gln Ile Gly Cys Gly Asn Val 405 410 415 Thr His Lys Phe Val Thr Leu His Lys Pro Lys Gln Gly Ala Asp Thr 420 425 430 Tyr Leu Glu Leu Gly Phe Lys Asn Gly Thr Leu Ala Pro Gly Ala Ser 435 440 445 Thr Gly Asn Ile Gln Leu Arg Leu His Asn Asp Asp Trp Ser Asn Tyr 450 455 460 Ala Gln Ser Gly Asp Tyr Ser Phe Phe Lys Ser Asn Thr Phe Lys Thr 465 470 475 480 Thr Lys Lys Ile Thr Leu Tyr Asp Gln Gly Lys Leu Ile Trp Gly Thr 485 490 495 Glu Pro Asn 17185PRTArtificial sequenceBacillus subtilis xylanase variant 17Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Phe Gly Gly Gly Ile Val 1 5 10 15 Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn 20 25 30 Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe 35 40 45 Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly 50 55 60 Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr 65 70 75 80 Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly 85 90 95 Thr Val Lys Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg 100 105 110 Tyr Asn Ala Pro Ser Ile Asp Gly Asp Asp Thr Thr Phe Thr Gln Tyr 115 120 125 Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile 130 135 140 Thr Phe Ser Asn His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu 145 150 155 160 Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Gln Ser 165 170 175 Ser Gly Ser Ser Asn Val Thr Val Trp 180 185 18185PRTArtificial sequenceBacillus subtilis xylanase variant 18Ala Ser Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Tyr Gly Ile Val 1 5 10 15 Asn Ala Val Asn Gly Ser Gly Gly Asn Tyr Ser Val Asn Trp Ser Asn 20 25 30 Thr Gly Asn Phe Val Val Gly Lys Gly Trp Thr Thr Gly Ser Pro Phe 35 40 45 Arg Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Asn Gly Asn Gly 50 55 60 Tyr Leu Thr Leu Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr 65 70 75 80 Val Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly 85 90 95 Thr Val Tyr Ser Asp Gly Gly Trp Tyr Asp Ile Tyr Thr Ala Thr Arg 100 105 110 Asp Asn Ala Pro Ser Ile Asp Gly Asp Phe Thr Thr Phe Thr Gln Tyr 115 120 125 Trp Ser Val Arg Gln Ser Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile 130 135 140 Thr Phe Ser Asn His Val Asn Ala Trp Arg Ser His Gly Met Asp Leu 145 150 155 160 Gly Ser Asn Trp Ala Tyr Gln Val Met Ala Thr Glu Gly Tyr Leu Ser 165 170 175 Ser Gly Ser Ser Asn Val Thr Val Trp 180 185

Claims

1. An enzyme exhibiting endo-1,4-.beta.-xylanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:17, and SEQ ID NO:18, or any functional fragment thereof.

2. The enzyme according to claim 1, which enzyme has a ratio in activity on soluble arabinoxylan substrate (WE-AX) to insoluble arabinoxylan substrate (WU-AX) arabinoxylan substrate of less than about 7.0, such as less than about 6.5, such as less than about 6.0, such as less than about 5.5, such as less than about 5.0, such as less than about 4.5.

3. An enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity, which enzyme comprises an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or any functional fragment thereof.

4. The enzyme according to claim 1, which enzyme has a temperature optimum in the range of 40-70.degree. C., such as in the range of 45-65.degree. C., such as in the range of 50-65.degree. C., such as in the range of 55-65.degree. C.

5. The enzyme according to claim 4, wherein said enzyme has at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

6. The enzyme according to claim 5 having a total number of amino acids of less than 350, such as less than 340, such as less than 330, such as less than 320, such as less than 310, such as less than 300 amino acids, such as in the range of 200 to 350, such as in the range of 220 to 345 amino acids.

7. The enzyme according to claim 6, wherein the amino acid sequence of said enzyme has at least one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions as compared to any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

8. The enzyme according to claim 7, wherein the amino acid sequence of said enzyme has a maximum of one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions compared to any one amino acid sequence selected from SEQ ID NO: 1-18, or any functional fragment thereof.

9. The enzyme according to claim 8, which enzyme comprises the amino acid sequence identified by any one of SEQ ID NO: 1-18, or any functional fragment thereof.

10. The enzyme according to claim 3, which enzyme consists of the amino acid sequence identified by any one of SEQ ID NO: 1-18, or any functional fragment thereof.

11. A DNA construct comprising a DNA sequence encoding an enzyme according to claim 1.

12. A recombinant expression vector comprising a DNA construct according to claim 11.

13. A cell that has been transformed with a DNA construct of claim 11.

14. A preparation comprising an enzyme according to claim 10.

15. A composition comprising an enzyme exhibiting endo-1,4-.beta.-xylanase activity according to claim 1 in combination with any one or more .beta.-glucanase.

16. The composition according to claim 15, wherein said one or more .beta.-glucanase is an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to any one of claims 3-10.

17. A composition comprising an enzyme exhibiting endo-1,3(4)-.beta.-glucanase activity according to claim 3 in combination with any one or more xylanase.

18. The composition according to claim 17, wherein said one or more xylanase is an enzyme exhibiting endo-1,4-.beta.-xylanase activity.

19. The composition according to claim 18, wherein said endo-1,3(4)-.beta.-glucanase activity and said endo-1,4-.beta.-xylanase activity are derived from at least two different enzymes, such as at least two different enzymes from two different species.

20. The composition according to claim 19, comprising a combination of at least two enzymes, said two enzymes, or two enzymes with an amino acid sequence having at least 80% sequence identity with the respective SEQ ID, or any functional fragment thereof, being selected from the list consisting of SEQ ID NO:1 and SEQ ID NO:7; SEQ ID NO:2 and SEQ ID NO:7; SEQ ID NO:3 and SEQ ID NO:7; SEQ ID NO:4 and SEQ ID NO:7; SEQ ID NO:5 and SEQ ID NO:7; SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:17 and SEQ ID NO:7; SEQ ID NO:18 and SEQ ID NO:7; SEQ ID NO:1 and SEQ ID NO:8; SEQ ID NO:2 and SEQ ID NO:8; SEQ ID NO:3 and SEQ ID NO:8; SEQ ID NO:4 and SEQ ID NO:8; SEQ ID NO:5 and SEQ ID NO:8; SEQ ID NO:6 and SEQ ID NO:8; SEQ ID NO:17 and SEQ ID NO:8; SEQ ID NO:18 and SEQ ID NO:8; SEQ ID NO:1 and SEQ ID NO:9; SEQ ID NO:2 and SEQ ID NO:9; SEQ ID NO:3 and SEQ ID NO:9; SEQ ID NO:4 and SEQ ID NO:9; SEQ ID NO:5 and SEQ ID NO:9; SEQ ID NO:6 and SEQ ID NO:9; SEQ ID NO:17 and SEQ ID NO:9; SEQ ID NO:18 and SEQ ID NO:9; SEQ ID NO:1 and SEQ ID NO:10; SEQ ID NO:2 and SEQ ID NO:10; SEQ ID NO:3 and SEQ ID NO:10; SEQ ID NO:4 and SEQ ID NO:10; SEQ ID NO:5 and SEQ ID NO:10; SEQ ID NO:6 and SEQ ID NO:10; SEQ ID NO:17 and SEQ ID NO:10; SEQ ID NO:18 and SEQ ID NO:10; SEQ ID NO:1 and SEQ ID NO:11; SEQ ID NO:2 and SEQ ID NO:11; SEQ ID NO:3 and SEQ ID NO:11; SEQ ID NO:4 and SEQ ID NO:11; SEQ ID NO:5 and SEQ ID NO:11; SEQ ID NO:6 and SEQ ID NO:11; SEQ ID NO:17 and SEQ ID NO:11; SEQ ID NO:18 and SEQ ID NO:11; SEQ ID NO:1 and SEQ ID NO:12; SEQ ID NO:2 and SEQ ID NO:12; SEQ ID NO:3 and SEQ ID NO:12; SEQ ID NO:4 and SEQ ID NO:12; SEQ ID NO:5 and SEQ ID NO:12; SEQ ID NO:6 and SEQ ID NO:12; SEQ ID NO:17 and SEQ ID NO:12; SEQ ID NO:18 and SEQ ID NO:12; SEQ ID NO:1 and SEQ ID NO:13; SEQ ID NO:2 and SEQ ID NO:13; SEQ ID NO:3 and SEQ ID NO:13; SEQ ID NO:4 and SEQ ID NO:13; SEQ ID NO:5 and SEQ ID NO:13; SEQ ID NO:6 and SEQ ID NO:13; SEQ ID NO:17 and SEQ ID NO:13; SEQ ID NO:18 and SEQ ID NO:13; SEQ ID NO:1 and SEQ ID NO:14; SEQ ID NO:2 and SEQ ID NO:14; SEQ ID NO:3 and SEQ ID NO:14; SEQ ID NO:4 and SEQ ID NO:14; SEQ ID NO:5 and SEQ ID NO:14; SEQ ID NO:6 and SEQ ID NO:14; SEQ ID NO:17 and SEQ ID NO:14; SEQ ID NO:18 and SEQ ID NO:14; SEQ ID NO:1 and SEQ ID NO:15; SEQ ID NO:2 and SEQ ID NO:15; SEQ ID NO:3 and SEQ ID NO:15; SEQ ID NO:4 and SEQ ID NO:15; SEQ ID NO:5 and SEQ ID NO:15; SEQ ID NO:6 and SEQ ID NO:15; SEQ ID NO:17 and SEQ ID NO:15; SEQ ID NO:18 and SEQ ID NO:15; SEQ ID NO:1 and SEQ ID NO:16; SEQ ID NO:2 and SEQ ID NO:16; SEQ ID NO:3 and SEQ ID NO:16; SEQ ID NO:4 and SEQ ID NO:16; SEQ ID NO:5 and SEQ ID NO:16; SEQ ID NO:6 and SEQ ID NO:16; SEQ ID NO:17 and SEQ ID NO:16; and SEQ ID NO:18 and SEQ ID NO:16.

21. The composition according to claim 20, wherein when used prior to the lautering in a brewing application the total pressure built up is reduced to a value of less than 470 mm WC, such as less than 450 mm WC, such as less than 430 mm WC, such as less than 410 mm WC, such as less than 390 mm WC, such as less than 370 mm WC, such as less than 350 mm WC, such as less than 330 mm WC, such as less than 310 mm WC, such as less than 300 mm WC, such as less than 290 mm WC.

22. The composition according to claim 21, wherein when used prior to the lautering in a brewing application the total pressure built up is reduced by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 or 95% compared to the use of a negative control without said composition.

23. The composition according to claim 22, wherein when used in a brewing application prior to the wort separation, the wort filterability as measured by volume wort collected after 5 min of filtration relative to a control without enzymes is increased to above 1.5, such as above 1.6, such as above 1.7, such as above 1.8, such as above 1.9, such as above 2.0, such as above 2.1, such as above 2.2, such as above 2.3, such as above 2.4, such as above 2.5.

24. The composition according to claim 23, when used in a brewing application prior to the wort separation, the wort filterability as measured by volume wort collected after 5 min of filtration is increased by at least 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300% as compared to the use of a negative control without said composition.

25. The composition according to claim 24, comprising any one or more further enzyme.

26. The composition according to claim 25, wherein said one or more further enzyme is selected from list consisting of a xylanase classified in EC 3.2.1.32, EC 3.2.1.136, or EC 3.2.1.156, a cellulase, a laminarinase, an endo-1,5-.alpha.-L-arabinanase, a beta-D-glucoside glucohydrolase, a .beta.-Xylosidase, a cellobiohydrolase, a glucan 1,4-beta-glucosidase, a xyloglucan-specific exo-beta-1,4-glucanase and an .alpha.-N-Arabinofuranosidase.

27. Use of an enzyme according to claim 1 in the production of a food, feed, or malt beverage product.

28. Use of an enzyme according to claim 1 in the production of dough or baked products.

29. Use of an enzyme according to claim 1 in the preparation of pulp or paper.

30. Use of an enzyme according to claim 1 for the preparation of cereal components.

31. The use according to claim 30, in which the cereal is rye, wheat, or barley.

32. Use of an enzyme according to claim 1 in the production of beer or modification of by-products from a brewing process.

33. Use of an enzyme according to claim 1 in the production of wine or juice.

34. Use of an enzyme according to claim 1 in the production of a first- or second-generation biofuel, such as bioethanol.

35. Method of altering filterability of a starch comprising material, said method comprising the step of treating said starch comprising material with enzyme according to claim 1.

36. Method of reducing pressure built up during lautering in a brewing application, said method comprising the step of treating a brewing mash with enzyme according to claim 1.

37. Method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, said method comprising the step of treating a starch comprising material with enzyme according to claim 1.

38. Method for the production of a brewing mash, said method comprising the step of treating a starch comprising material with enzyme according to claim 1.

39. Method for the production of a first- or second-generation biofuel, such as bioethanol, said method comprising the step of treating a starch comprising material with an enzyme according to claim 1.

40. Product obtained by the method according to claim 39.

41. A composition comprising the product according to claim 40, such as wherein the product is in a range of 0.1%-99.9%.