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Xylanases, Nucleic Acids Encoding Them and Methods for Making and Using Them

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Xylanases, Nucleic Acids Encoding Them and Methods for Making and Using Them (19) TZZ _T (11) EP 2 548 954 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 23.01.2013 Bulletin 2013/04 C12N 9/24 (2006.01) (21) Application number: 12168120.9 (22) Date of filing: 14.02.2007 (84) Designated Contracting States: • Todaro, Thomas AT BE BG CH CY CZ DE DK EE ES FI FR GB GR San Diego, CA 92110 (US) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Hazlewood, Geoff SK TR Newbury, Berkshire RG14 2RS (GB) • Chang, Kristine (30) Priority: 14.02.2006 US 773122 P San Diego, CA 92122 (US) • Chang, Hwai (62) Document number(s) of the earlier application(s) in San Marcos, CA 92069 (US) accordance with Art. 76 EPC: • Desantis, Grace 07751203.6 / 1 989 302 San Diego, CA 92129 (US) (71) Applicant: Verenium Corporation (74) Representative: Wakerley, Helen Rachael San Diego, CA 92121 (US) Reddie & Grose LLP 16 Theobalds Road (72) Inventors: London WC1X 8PL (GB) • Weiner, David Del Mar, CA 92014 (US) Remarks: • Hansen, Connie Jo •Thecomplete document including Reference Tables San Diego, CA 92115 (US) and the Sequence Listing can be downloaded from • Hancock, Charles the EPO website San Marcos, CA 92078 (US) •This application was filed on 15-05-2012 as a • Woodward, Thomas divisional application to the application mentioned Scottsville, VA 24590 (US) under INID code 62. • Beaver, Scott W •Claims filed after the date of filing of the application/ San Diego, CA 92078 (US) after the date of receipt of the divisional application • Blum, David (Rule 68(4) EPC). Nashville, TN 37205 (US) •A request for correction of the sequence listing has • Healey, Shaun been filed pursuant to Rule 139 EPC. A decision on Carlsbad, CA 92008 (US) the request will be taken during the proceedings • Varvak, Alexander before the Examining Division (Guidelines for San Diego, CA 92103 (US) Examination in the EPO, A-V, 3.). (54) Xylanases, nucleic acids encoding them and methods for making and using them (57) The invention relates to enzymes having xyla- hydrolyzing hemicelluloses in any plant or wood or wood nase, mannanase and/or glucanase activity, e.g., cata- product, wood waste, paper pulp, paper product or paper lyzing hydrolysis of internal β-1,4-xylosidic linkages or waste or byproduct. In addition, methods of designing endo- β-1,4-glucanase linkages; and/or degrading a lin- new xylanases, mannanases and/or glucanases and ear polysaccharide beta-1,4-xylan into xylose. Thus, the methods of use thereof are also provided. The xylanases, invention provides methods and processes for breaking mannanases and/or glucanases have increased activity down hemicellulose, which is a major component of the and stability at increased pH and temperature. cell wall of plants, including methods and processes for EP 2 548 954 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 548 954 A1 Description [0001] The content of the following submission on compact discs is incorporated herein by reference in its entirety: A computer readable form (CRF) of the Sequence Listing on compact disc (file name: 564462007967, date recorded: 5 February 14, 2007, size: 1,851,392 bytes); a triplicate compact disc copy of the Sequence Listing (COPY 1) (file name: 564462007967, date recorded: February 14, 2007, size: 1,851,392 bytes); a triplicate compact disc copy of the Sequence Listing (COPY 2) (file name: 564462007967, date recorded: February 14, 2007, size: 1,851,392 bytes); and a triplicate compact disc copy of the Sequence Listing (COPY 3) (file name: 564462007967, date recorded: February 14, 2007, size: 1,851,392 bytes). 10 FIELD OF THE INVENTION [0002] This invention relates generally to enzymes, polynucleotides encoding the enzymes, the use of such polynu- cleotides and polypeptides and more specifically to enzymes having xylanase activity, e.g., catalyzing hydrolysis of 15 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; or, a glucanase activity, e.g., an endoglucanase activity, for example, catalyzing hydrolysis of internal endo- β-1,4- and/or 1,3- glucanase linkages, a xylanase activity, and/or a mannanase activity. 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 organic compound, plant or wood or wood product or 20 byproduct, wood waste, paper pulp, paper product or paper waste or byproduct. BACKGROUND [0003] Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.2.1.8) hydrolyze internal β-1,4-xylosidic linkages in xylan to 25 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 paper production. Xylanases are formed by fungi and bacteria. [0004] Arabinoxylans are major non-starch polysaccharides of cereals representing 2.5 - 7.1% w/w depending on 30 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, 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 35 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 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- 40 version efficiencies. [0005] Xylanases have been shown to be useful in biobleaching and treatment of chemical pulps (see, for example, U.S. Pat. No. 5,202,249), biobleaching and treatment of wood or paper pulps (see, for example, U.S. Pat. Nos. 5,179,021, 5,116,746, 5,407,827, 5,405,769, 5,395,765, 5,369,024, 5,457,045, 5,434,071, 5,498,534, 5,591,304, 5,645,686, 5,725,732, 5,759,840, 5,834,301, 5,871,730 and 6,057,438) in reducing lignin in wood and modifying wood (see, for 45 example, U.S. Pat. Nos. 5,486,468 and 5,770,012) as flour, dough and bread improvers (see, for example, U.S. Pat. Nos. 5,108,765 and 5,306,633) as feed additives and/or supplements, as set forth above (see, for example, U.S. Pat. Nos. 5,432,074, 5,429,828, 5,612,055, 5,720,971, 5,981,233, 5,948,667, 6,099,844, 6,132,727 and 6,132,716), in man- ufacturing cellulose solutions (see, for example, U.S. Pat. No.5,760,211). Detergent compositions having xylanase activity are used for fruit, vegetables and/or mud and clay compounds (see, for example, U.S. Pat. No. 5,786,316). 50 Xylanases may also be used in hydrolysis of hemicellulose for which it is selective, particularly in the presence of cellulose. Additionally, the cellulase rich retentate is suitable for the hydrolysis of cellulose (see, for example, U.S. Pat. No. 4,725,544). [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 55 useful in the paper and pulp industry would decrease the need for bleaching chemicals, such as chlorine dioxide. 2 EP 2 548 954 A1 SUMMARY OF THE INVENTION [0007] The invention provides enzymes having: xylanase activity, e.g., catalyzing hydrolysis of internal β-1,4-xylosidic linkages or endo- β-1,4-glucanase linkages; and/or, having a glucanase activity, e.g., an endoglucanase activity, for 5 example, catalyzing hydrolysis of internal endo-β -1,4- and/or 1,3-glucanase linkages, a xylanase activity, and/or a mannanase activity; and, nucleic acids encoding them, vectors and cells comprising them, probes for amplifying and identifying these xylanase-encoding nucleic acids, and methods for making and using these polypeptides and peptides. [0008] For example, the invention provides enzymes having xylanase activity, and compositions and methods com- prising them, for hydrolyzing internal β-1,4-xylosidic linkages or endo- β-1,4-glucanase linkages, or hemicelluloses, in 10 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, which is a major component of the cell wall of plants. [0009] In one aspect, the glucanase activity of a polypeptide or peptide of the invention (which includes a protein or 15 peptide encoded by a nucleic acid of the invention) comprises an endoglucanase activity, e.g., endo-1,4- and/or 1,3- beta-D-glucan 4-glucano hydrolase activity.
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