USOO9238806B2 (12) United States Patent (10) Patent No.: US 9.238,806 B2 Hill et al. (45) Date of Patent: Jan. 19, 2016 (54) CELLULASE ENZYME MIXTURES FOR 6,071,735 A 6/2000 Schulein et al. DEPILLING AND USES THEREOF : 29 A 358 SE et 1 k --- U.S. ca. 6,162,782 A 12/2000 Clarkson et al. (75) Inventors: Christopher Hill, Nepean (CA); John J. 6,268, 196 B1 7/2001 Fowler et al. Tomashek, Ottawa (CA); Loreta 6,458,955 B1 10/2002 Gattuso Gudynaite-Savitch, Kanata (CA); 6,723,549 B2 4/2004 Miettinen-Oinonen et al. Guylaine Therrien, Gatineau (CA); E. 2.39 Still et al Sophie Calixte, Ottawa (CA) - 4 J. akamura et al. s 7,041,488 B2 5/2006 Outtrup et al. 7,049,125 B2 5/2006 Dunn-Coleman et al. (73) Assignee: Novozymes A/S, Bagsvaerd (DK) 7,138,261 B2 11/2006 Nakane et al. 7,226,773 B2 6/2007 Schulein et al. (*) Notice: Subject to any disclaimer, the term of this 7.256,032 B2 8/2007 Valtakari et al. patent is extended or adjusted under 35 223 R: 258. stoponen et al. U.S.C. 154(b) by 0 days. 7,445,922- B2 11/2008 Nakanea et Ca. al. (21) Appl. N 13A981,198 7,449,318 B2 11/2008 Jones et al. ppl. No.: 9 7,741,093 B2 6/2010 Vehmaanpera et al. 7,785,854 B2 * 8/2010 St-Pierre et al. .............. 435/200 (22) PCT Filed: Feb. 9, 2012 8,043,828 B2 * 10/2011 Bodie et al. ..................... 435/18 8,044.264 B2 * 10/2011 Lopez de Leon et al. ... 800/288 8,609,387 B2 * 12/2013 Valtakari et al. .............. 435,209 (86). PCT No.: PCT/CA2O12/OSOOT4 8,609,388 B2 * 12/2013 Masri et al. ................... 435,209 S371 (c)(1), (Continued) (2), (4) Date: Aug. 28, 2013 FOREIGN PATENT DOCUMENTS (87) PCT Pub. No.: WO2012/106824 EP O 866 165 B2 8, 2003 PCT Pub. Date: Aug. 16, 2012 WO 92,17574 A1 10, 1992 (65) Prior Publication Data (Continued) OTHER PUBLICATIONS US 2013/03375.42 A1 Dec. 19, 2013 Broun et al., Catalytic plasticity of fatty acid modification enzymes Related U.S. Application Data underlying chemical diversity of plant lipids. Science, 1998, vol. 282: AV 1315-1317. (60) Provisional application No. 61/440,985, filed on Feb. Chica et al., Semi-rational approaches to engineering enzyme activ 9, 2011. ity: combining the benefits of directed evolution and rational design. Curr. Opi. Biotechnol., 2005, vol. 16: 378-384.* (51) Int. Cl. Devos et al., Practical limits of function prediction. Proteins: Struc CI2N 9/42 (2006.01) ture, Function, and Genetics. 2000, vol. 41: 98-107. CI2N I/00 (2006.01) Seffernicket al., Melamine deaminase and Atrazine chlorohydrolase: D06M 16/00 (2006.01) 98 percent identical but functionally different. J. Bacteriol., 2001, C07K L/00 2006.O1 vol. 183 (8): 2405-2410.* C7H 2L/04 30 6 8: Sen et al., Developments in directed evolution for improving enzyme 52) U.S. C functions. Appl. Biochem. Biotechnol., 2007, vol. 143: 212-223.* (52) AV e. we Whisstock et al., Prediction of protein function from protein CPC ....... CI2N 9/2437 (2013.01); C12Y302/01004 sequence. Q. Rev. Biophysics., 2003, vol. 36 (3): 307-340.* (2013.01); C12Y302/01091 (2013.01): D06M Continued 16/00 (2013.01): D06M 16/003 (2013.01) (Continued) (58) Field of Classification Search Primary Examiner — Ganapathirama Raghu CPC. C12N 9/2437; D06M 16/00: D06M 16/003; (74) Attorney, Agent, or Firm — Elias Lambiris C12Y 302/01004; C12Y 302/01091 57 ABSTRACT USPC ............. 435/209, 254.6, 263: 536/23.1, 23.2: (57) - - - - 530/350 The present invention relates to a depilling composition com See application file for complete search history. prising an enzyme mixture that comprises a Family 45 cellu lase and one or more additional cellulases selected from a (56) References Cited Family 5 cellulase, a Family 6 cellulase or a combination thereof. The enzyme mixture may be characterized in that the U.S. PATENT DOCUMENTS Family 45 and Family 5 cellulases or the Family 45 and 5,213,581 A 5, 1993 Olson et al. Family 6 cellulases are present at a weight ratio that exhibits 5,691,178 A 11/1997 Shulein et al. synergy in an assay that measures specific depilling activity. 5,792,641 A * 8/1998 Schulein et al. .............. 435,209 The enzyme mixture may be secreted by a genetically modi 5,811,381 A 9, 1998 Emalfarb et al. 5,866.407 A * 2/1999 Foody et al. .................. 435.263 fied microbe overexpressing the foregoing cellulases. Also 5,874.293 A 2, 1999 Miettinen-Oinonen et al. provided is a process for depilling that comprises a step of 5,916,798 A 6, 1999 Lund et al. contacting cellulose-containing goods with the depilling 5.948,672 A 9, 1999 Rasmussen et al. composition. 5,958,083. A 9, 1999 Onishi et al. 6,001,639 A 12/1999 Schulein et al. 19 Claims, 31 Drawing Sheets US 9.238,806 B2 Page 2 (56) References Cited Herpoel-Gimbert etal, Biotechnology for Biofuels, pp. 1-12 (2008). Hetti Palonen, VTT Publications 520, pp. 1-84 (2004). U.S. PATENT DOCUMENTS Igarashietal. 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