(19) TZZ Z_T (11) EP 2 205 744 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/52 (2006.01) C12N 15/09 (2006.01) 14.01.2015 Bulletin 2015/03 C12N 9/42 (2006.01) (21) Application number: 08797071.1 (86) International application number: PCT/US2008/072030 (22) Date of filing: 01.08.2008 (87) International publication number: WO 2009/045627 (09.04.2009 Gazette 2009/15) (54) XYLANASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING AND USING THEM XYLENASEN, DAFÜR CODIERENDE NUKLEINSÄUREN UND VERFAHREN ZUR HERSTELLUNG UND VERWENDUNG DAVON XYLANASES, ACIDES NUCLÉIQUES CODANT POUR ELLES ET LEURS MÉTHODES D’OBTENTION ET D’UTILISATION (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A2-2007/095398 US-B1- 6 365 390 HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR • ARASE AKEMI ET AL: "Stabilization of xylanase by random mutagenesis" FEBS LETTERS, (30) Priority: 03.10.2007 US 977348 P ELSEVIER, AMSTERDAM, NL LNKD- DOI: 10.1016/0014-5793(93)81199-A, vol. 316, no. 2, 1 (43) Date of publication of application: January 1993 (1993-01-01), pages 123-127, 14.07.2010 Bulletin 2010/28 XP002196471 ISSN: 0014-5793 • ANDREWS SIMON R ET AL: "The use of forced (60) Divisional application: protein evolution to investigate and improve 13181497.2 / 2 708 602 stability of family 10 xylanases - The production of Ca2+-independent stable xylanases" (73) Proprietor: BP Corporation North America Inc. JOURNAL OF BIOLOGICAL CHEMISTRY, vol. Houston, TX 77079 (US) 279, no. 52, 24 December 2004 (2004-12-24), pages 54369-54379, XP002591513 ISSN: (72) Inventors: 0021-9258 • GRAY, Kevin, A. • DATABASE NCBI 26 April 1993 XP008135720 San Diego, CA 92121 (US) Database accession no. M22759 • DIRMEIER, Reinhard • GREPINET, OLIVIER ET AL.: ’Nucleotide San Diego, CA 92121 (US) sequence and deletion analysis of the xylanase gene of Clostridium thermocellum.’ J (74) Representative: Wakerley, Helen Rachael BACTERIOL. vol. 170, no. 10, October 1988, Reddie & Grose LLP pages 4582 - 8, XP000874276 16 Theobalds Road • DATABASE NCBI 09 December 2004 London WC1X 8PL (GB) XP008135721 Database accession no. AY502070 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 205 744 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 205 744 B1 Description [0001] This application claims benefit of provisional application US Serial No. 60/977,348 filed 03 October 2007. [0002] This application was filed electronically via the USPTO EFS-WEB server, as authorized and set forth in MPEP 5 § 1730 II.B.2.(a)(A), and this electronic filing includes an electronically submitted sequence (SEQ ID) listing; the entire content of this sequence listing is herein incorporated for all purposes. The sequence listing is identified on the elec- tronically filed .txt file as follows: File Name Date of Creation Size 10 564462016140.txt August 1, 2008 88,832 bytes FIELD OF THE INVENTION 15 [0003] This invention relates generally to enzymes, polynucleotides encoding the enzymes, the use of such polynu- cleotides and polypeptides, the enzymes having xylanase activity, e.g., endoxylanase activity, and/or catalyzing hydrol- ysis of internal β-1,4-xylosidic linkages or endo- β-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any 20 organic compound, plant or wood or wood product or byproduct, wood waste, paper pulp, paper product or paper waste or byproduct. The invention further provides methods and processes for breaking down plant matter containing cellulose and/or hemicellulose into simple sugars using the "cocktails" of the invention. BACKGROUND 25 [0004] Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.2.1.8) hydrolyze internal β-1,4-xylosidic linkages in xylan to produce smaller molecular weight xylose and xylo-oligomers. Xylans are polysaccharides formed from 1,4- β-glycoside- linked D-xylopyranoses. Xylanases are of considerable commercial value, being used in the food industry, for baking and fruit and vegetable processing, breakdown of agricultural waste, in the manufacture of animal feed and in pulp and 30 paper production. Xylanases are formed by fungi and bacteria. [0005] Arabinoxylans are major non-starch polysaccharides of cereals representing 2.5 - 7.1% w/w depending on variety and growth conditions. The physicochemical properties of this polysaccharide are such that it gives rise to viscous solutions or even gels under oxidative conditions. In addition, arabinoxylans have high water-binding capacity and may have a role in protein foam stability. All of these characteristics present problems for several industries including brewing, 35 baking, animal nutrition and paper manufacturing. In brewing applications, the presence of xylan results in wort filterability and haze formation issues. In baking applications (especially for cookies and crackers), these arabinoxylans create sticky doughs that are difficult to machine and reduce biscuit size. In addition, this carbohydrate is implicated in rapid rehydration of the baked product resulting in loss of crispiness and reduced shelf-life. For monogastric animal feed applications with cereal diets, arabinoxylan is a major contributing factor to viscosity of gut contents and thereby adversely 40 affects the digestibility of the feed and animal growth rate. For ruminant animals, these polysaccharides represent substantial components of fiber intake and more complete digestion of arabinoxylans would facilitate higher feed con- version efficiencies. [0006] There remains a need in the art for xylanases to be used in the paper and pulp industry, for example, where the enzyme is active in the temperature range of 65°C to 75°C and at a pH of approximately 10. Additionally, an enzyme 45 useful in the paper and pulp industry would decrease the need for bleaching chemicals, such as chlorine dioxide. [0007] Additionally, there remains a need to provide efficient, low cost processes and compositions for producing bioalcohols, biofuels and/or biofuel- (e.g., bioethanol-, propanol-, butanol- and/or methanol-) by conversion of biomass. An enzyme or enzyme "cocktail" could provide a route to convert biomass into sugars that could then be fermented into biofuels. 50 SUMMARY OF THE INVENTION [0008] The invention provides enzymes having: xylanase activity, e.g., endoxylanase activity, and/or catalyzing hy- drolysis of internal β-1,4-xylosidic linkages or endo- β-1,4-glucanase linkages; and, nucleic acids encoding them, vectors 55 and cells comprising them, probes for amplifying and identifying these xylanase-encoding nucleic acids, and methods for making and using these polypeptides and peptides. [0009] For example, the invention provides enzymes having xylanase (e.g., endoxylanase activity), and compositions 2 EP 2 205 744 B1 and methods comprising them, for hydrolyzing internal β-1,4-xylosidic linkages or endo- β-1,4-glucanase linkages, or hemicelluloses, in a wood, wood product, paper pulp, paper product or paper waste. In one aspect, the xylanase activity comprises catalyzing hydrolysis of xylan, e.g., degrading a linear polysaccharide beta-1,4-xylan into a xylose. Thus, the invention provides methods and processes for breaking down a xylan-comprising composition and/or a hemicellulose, 5 which is a major component of the cell wall of plants. [0010] In one aspect, the xylanase activity comprises hydrolyzing a glucan or other polysaccharide to produce a smaller molecular weight polysaccharide or oligomer. In one aspect, the glucan comprises a beta-glucan, such as a water soluble beta-glucan. The invention provides enzymes, compositions, methods and processes for hydrolyzing hemicelluloses in any organic matter, including cells, plants and/or wood or wood products, wood waste, paper pulp, paper products or 10 paper waste or byproducts. The invention further provides methods and processes for breaking down plant matter containing cellulose and/or hemicellulose into simple sugars using the "cocktails" of the invention. [0011] The invention provides enzymes for the bioconversion of any biomass, e.g., a lignocellulosic residue, into fermentable sugars or polysaccharides; and these sugars or polysaccharides can be used as a chemical feedstock for the production of alcohols such as ethanol, propanol, butanol and/or methanol, production of fuels, e.g., biofuels such 15 as synthetic liquids or gases, such as syngas, and the production of other fermentation products, e.g. succinic acid, lactic acid, or acetic acid. Enzymes of the invention can be added to bioconversion and other industrial processes continuously, in batches or by fed-batch methods. In another aspect, enzymes of the invention can be recycled in bioconversion and other industrial processes, thereby lowering enzyme requirements. [0012] In one aspect, the enzymes of the invention have an increased catalytic rate to improve the process of substrate 20 (e.g., a lignocellulosic residue, cellulose, bagasse) hydrolysis. This increased efficiency in catalytic rate leads to an increased efficiency in producing sugars or polysaccharides, which can be useful in industrial, agricultural or medical applications, e.g., to make a biofuel or an alcohol such as ethanol, propanol, butanol and/or methanol. In one aspect, sugars produced by hydrolysis using enzymes of this invention can be used by microorganisms for alcohol (e.g., ethanol, propanol, butanol and/or methanol) production and/or fuel (e.g., biofuel) production.