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. Applied and Environmental Microbiology, vol. 74, No. 18, pp. 5628-5634 (2008). 8,679,791 B2* 3/2014 Festersen et al...... 435.93 Jahangeer et al. Screening and Characterization of Fungal Cellulases, 2003/0054539 A1 3/2003 Schulein et al. vol. 37, No. 3, pp. 739-748 (2005). 2003/0129723 A1 7/2003 Ishikawa et al. Kogaetal. Applied and Environmental Microbiology, vol. 74, No. 13, 2007/0111278 A1 5/2007 Koga et al. pp. 4210-4217 (2008). Matthew etal, Journal of Scientific and Industrial Research, vol. 67, FOREIGN PATENT DOCUMENTS pp. 898-907 (2008). Mats Sandgren, Structural and Functional Studies of Glycoside WO 97.14804 A1 4f1997 Hydrolase Family, vol. 12, pp. 1-70 (2003). WO 2007/057418 A1 5/2007 WO 2007/071820 A1 6, 2007 Miettinen-Oionen, VTT Publications 550, pp. 1-153 (2004). WO 2007.118935 A1 10/2007 MK Bhat, Biotechnology Advances, vol. 18, pp. 355-383 (2000). WO 2007/147264 A1 12/2007 Murashima et al. Biosci. Biotechnol. Biochem, vol. 70, No. 9, pp. WO 2008/088724 A2 T 2008 2205-2212 (2006). WO 2010/076388 A1 T 2010 Pere et al., J. Biotechnol, vol. 89, No. 2-3, pp. 247-255 (2001). Ramos et al. Biocatalysis and Biotransformation, vol. 25, No. 1, pp. OTHER PUBLICATIONS 35-42 (2007). Rosgaard et al. Biotechnol. Prog. vol. 23, pp. 1270-1276 (2007). Witkowski et al., Conversion of b-ketoacyl synthase to a Malonyl Sarah Teter and Joel Cherry, Improving Cellulose Hydrolysis, 1-7 Decarboxylase by replacement of the active cysteine with glutamine. (2001). Biochemistry, 1999, vol. 38: 11643-1 1650.* Shimonaka et al. Biosci Biotechnol Biochem, vol. 70, No. 4, pp. Boisset et al., Biotechnology and Bioengineering, vol. 72, No. 3, pp. 1013-1016 (2006). 339-345 (2001). Shimonaka et al. Biosci Biotechnol Biochem, vol. 70, No. 10, pp. Gusakov et al., Biotechnology and Bioengineering, vol.97, No. 5, pp. 2460-2466 (2006). 1028-1038 (2007). Cavaco-Paulo, The Treatment of Cotton Cellulose with Miettinen-Oinonen et al., Applied and Environmental Microbiology, Thrichoderma Reesei Engineered Cellulases, pp. 227-234 (1998). vol. 68, No. 8, pp. 3956-3964 (2002). Szijarto et al. Journal of Biotechnology, vol. 136, 140-147 (2008). Andersen et al. Enzyme and Microbiol Technology, vol. 42, pp. Szijarto N. Thesis of Applied Biotechnology, pp. 1-12 (2007). 362-370 (2008). Pritt Valjamae. The Kinectics of Cellulose Enzymatic Hydrolysis, pp. Andersen J. Enzymatic Hyrdrolysis of Cellulose, pp. 1-162—(2007). 1-55 (2002). Anu Nutt, Hydrolytic and Oxidative Mechanisms Involved in Cellu Tamaru et al. Environmental Technology, vol. 31, No. 8.9, pp. 889 lose Degradation, 1-52 (2006). 903 (2010). Azevedo et al. Enzyme and Microbiol Technology, vol. 27, pp. 325 Viviana Kopcke, pp. 1-63 (2008). 329 (2000). Vlasenko et al. Bioresource Technology, vol. 101, pp. 2405-2411 Azevedo et al. Applied Biochemistry and Biotechnology, vol. 101, (2010). pp. 61-76 (2002). Zhang et al. Biotechnol Bioeng, vol. 88, No. 7, pp. 797-824 (2004). Cavaco-Paulo et al. Textile Research Journal, vol. 68, No. 6, pp. Zhang et al. Biotechnology Advances, vol. 24, pp. 452-481 (2006). 398-401 (1998). Tzanov et al. Journal of Biotechnology, vol. 6, No. 3 pp. 146-154 Cavaco-Paulo et al. Textile Research Journal, vol. 68, No. 4, pp. (2003). 273-280 (1998). Cavaco-Paulo Almeida, Textile Research Journal, vol. 66, No. 5, pp. Cavaco-Paulo et al. Enzyme Technology, pp. 28-32 (1996). 287-294 (1996). Cavaco-Paulo, Carbohydrate Polymers, vol. 37, pp. 273-277 (1998). Cavaco-Paulo et al., Textile Chemist and Colorist, pp. 28-32 (Jun. Christopher French, Journal of the Royal Society, pp. 1-13 (2009). 1996). Dashtban et al. International Journal of Biological Sciences, vol. 5, Miettinen-Oinonen et al., Journal of Biotechnology, vol. 116, No. 3, No. 6, pp. 578-595 (2009). pp. 305-317 (2005). Eberhardt et al. Microbiology, vol. 146, pp. 1999-2008 (2000). Yamada et al., Biosci. Biotechnol. Biochem... vol. 60, No. 1, pp. 45-50 Enebro et al. Cellulose, vol. 16, pp. 271-280 (2009). (2005). Heikinheimo et al. Textile Research Journal, vol. 71, No. 8, pp. Chhavi et al., The Proteins Behind Fuzz Removal, pp. 1-5 (2007). 672-677 (2001). Heikinheimo, VTT Publication 483, 1-82 (2002). * cited by examiner

U.S. Patent Jan. 19, 2016 Sheet 16 of 31 US 9.238,806 B2

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Figure 7 U.S. Patent Jan. 19, 2016 Sheet 20 of 31 US 9.238,806 B2

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Figure 8 U.S. Patent Jan. 19, 2016 Sheet 21 of 31 US 9.238,806 B2

SEQ ID NO 3 83 SGTATYSGNPFWG------WTPWANAYYASEVSSLAIP 114 SEQ ID NO : 79 - - -ATYSGNPFWG------WTPWANAYYASEWSSIAIP SEQ ID NO : 8O - - -ATYSGNPFWG------WTPWANAYYASEWSSLAIP SEQ ID NO : 81 - - -ATYSGNPFWG------WTPWANAYYASEWSSILAIP SEQ ID NO : 82 - - -ATYSGNPFVG------WTPWANAYYASEVSSILAIP SEQ ID NO : 83 - - -ATYSGNPFVG------WTPWANAYYASEVSSLAIP SEQ ID NO : 84 - - -ATASGNPESG------YOLYVNPYYSSEVOSIAIP SEQ ID NO : 85 - - -ASASGNPFSG------YOLYVNPYYSSEVASLAIP SEQ ID NO : 86 - - -ATAGGNPEEG ------YDLYWNPYYKSEVESLAIP SEQ ID NO : 87 - - -ASATGNPFEG------YQLYANPYYKSQVESSAIP SEQ ID NO : 88 - - -AAASGNPFSG------YQLYANPYYSSEVHTLAIP SEQ ID NO : 89 - - -ASASGNPFEG------YQLYANPYYASEVISLAIP SEQ ID NO : 90 ---PVATNNPFSG------VDLWANNYYRSEVSTLAIP SEQ ID NO : 91. - - -PAASDNPYAG------VDLWANNYYRSEWMNLAVP SEQ ID NO : 92 - - -ASFTGNPFLG ------VQGWANSYYSSEIYNHAIP SEQ ID NO : 93 ---VQATGNPFEG------YQLYANPYYSSEVMTLAVP SEQ ID NO: 94 - - -ASATGNPFEG------YQLYVNPYYKSQVESSAIP SEQ ID NO : 95 - - -ASFTGNPFAG------WNLFPNKFYSSEVHTLAIP SEQ D NO: 96 - - -ASYNGNPFSG------VQLWANTYYSSEVHTLAIP SEQ ID NO 97 - - -ASYNGNPESG------VQLWANTYYSSEVHTLAIP SEQ ID NO : 98 - - -ATYTGNPFLG ------VNQWANNFYRSEIMNIAVP SEQ ID NO : 99 - - -ASYNGNPFEG------VQLWANNYYRSEVHTLAIP SEQ ID NO : 1OO GNPFEG------VQLWANNYYRSEVHTLAIP SEQ ID NO: 101 - - -AAPSGNPFAG------KNEYANPYYSSEWHTLAMP SEQ ID NO : 102 AGNPYTG------KTVWLSPFYADEVAQAAAD SEQ ID NO : 103 ---TPAAGNPEVG------WTPFISPYYAAEVAAAADA SEQ ID NO : 104 ---TPAAGNPFTG------YEIYLSPYYANEIAAAVTQ SEQ ID NO : 105 ---TPAAGNPFT------EQIYLSPYYANEIAAAVTQ SEQ ID NO : 106 ---QANSSNPFAG------HTIYPNPYYSNEIDEFAIP SEQ ID NO : 107 ---PPSANNPWTG------FOIFLSPYYANEVAAAAKO SEQ ID NO : 108 - - -WPAAGNPYTG------YEIYLSPYYAAEAQAAAAQ SEQ ID NO : 109 ---LDASTNVFOQ------YTILHPNNYRAEVEAAAEA SEQ ID NO : 11 O ---TPDAGNPYIGYDVSHVLWCQIYLSPYYADEVAAAVSA SEQ ID NO : 111 ---TPAAGNPFTG------FOVYLSPYYSAEIASAAAA SEQ ID NO : 112 ---LDASTNWWKK------YTILHANKFYRTEVEAAWAA SEQ ID NO: 113 ---LDASTNWFSK------YTILHPNSFYRAEWEAAAEA

Figure 9A

U.S. Patent Jan. 19, 2016 Sheet 23 of 31 US 9.238,806 B2

SEQ ID NO:3 151 DIRTANKNGGN--YAGQFVVYDLPDRDCAALASNGEYSIA SEQ ID NO: 79 DIRTANKNGGN--YAGQFVVYDLPDRDCAALASNGEYSIA SEQ ID NO: 80 DIRTANKNGGN--YAGOFVVYDLPDRDCAALASNGEYSIA SEQ ID NO: 81 DIRTANKNGGN--YAGQFVVYDLPDRDCAALASNGEYSIA SEO ID NO: 82 DIRTANKNGGN--YAGOFVVYDLPDRDCAALASNGEYSIA SEQ ID NO: 83 DIRTANKNGGN--YAGQFVVYDLPDRDCAALASNGEYSIA SEQ ID NO: 84 DIRSQNAAGANPPIAGQFVVYDLPDRDCAALASNGEFAIS SEO ID NO: 85 DIQSQNAAGANPPIAGOFVVYDLPDRDCAALASNGEYSIA SEQ ID NO: 86 DKKNAAGANPPTAGFWWYDLPDRDCAAASNGEFS SEQ ID NO: 87 DIQTQNAAGASPPIAGIFVVYDLPDRDCSALASNGEYSIS SEO ID NO: 88 NEAANKAGASPPAGIFVVYDLPDRDCAAAASNGEYTVA SEQ ID NO: 89 DIQSQNAAGADPPIAGIFVVYDLPDRDCAAAASNGEFSIA SEQ ID NO: 90 DIRKANKAGGN--YAGQFVVYDLPDRDCAAAASNGEYSLD SEQ ID NO: 91 DIRKANKAGGK--YAGOFVVYDLPNRDCAAAASNGEYSLD SEQ ID NO: 92 ERAANKAGANPPYAAHEWWYDLPDRDCAAAASNGEFSA. SEQ ID NO: 93 DIKEQNDAGANPPIAGIFVVYNLPDRDCAALASNGELSIA SEQ ID NO : 94 DIOTONAAGANPPIAGIFVVYDLPDRDCAALASNGEYAIS SEQ ID NO: 95 DVRAANAAGGN--YAAQLVVYDLPDRDCAAAASNGEFSIA SEQ ID NO: 96 EIRAANQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIA SEQ ID NO: 97 EIRAANQRGANPPYAGIFVVYDLPDRDCAAAASNGEWSIA SEQ ID NO: 98 DVRAANARGGN-- YASILVVYNLPDRDCAAAASNGEFAA SEO ID NO: 99 EIRAANOAGANPPYAAQIVVYDLPDRDCAAAASNGEWAIA SEQ ID NO:100 EIREANQAGANPQYAAQIVVYDLPDRDCAAAASNGEWAIA SEQ ID NO: 101 DIKAKNAAGAN--LMGTFWWYDLPDRDCAALASNGELKID SEO ID NO: 102 DAR------SKN-QLVOIVVYDLPDRDCAALASNGEFSLA SEQ ID NO : 103 DASALOKSSGOP-OVVOIVVYDLPDRDCAAKASNGEFSIA SEQ ID NO: 104 DASAKQKSEGKN-YLVOIVVYDLPDRDCAALASNGEFTIA SEQ ID NO : 105 DASAKQKSEGKN-YLVOIVVYDLPDRDCAALASNGEFTIA SEQ ID NO: 106 GIQEANAAGQNPPYIGAIVVYDLPNRDCAAAASNGEFSLE SEQ ID NO: 107 SASALGKSTGTK-QLVQIVIYDLPDRDCAAKASNGEFSIA SEQ ID NO : 108 EASALGKSSGKK-YLVOIVVYDLPDRDCAALASNGEFSIA SEQ ID NO: 109 DWPCENVG------WYDLPGRDCAAKASNGELKWG SEQ ID NO: 110 DALSIQQSTGRN-QLVQIVVYDLPDRDCAALASNGEFSIA SEQ ID NO: 111 DASSIOTKTGOK-QLVPIVVYELPDRDCAAKASNGEFSIA SEQ ID NO: 112 DVPCDHILG------LVIYDLPGRDCAAKASNGELAWG SEO ID NO: 113 DQPCDHILG------LVYDLPGRDCAAKASNGELKVG

Figure 9C U.S. Patent Jan. 19, 2016 Sheet 24 of 31 US 9.238,806 B2

SEQ ID NO:3 189 DGGVAKYK-NYIDTIRQIVV------EY 209 SEQ ID NO: 79 DGGVAKYK-NYIDTIRQIVV------EY SEQ ID NO: 80 DGGVAKYK-NYIDTIRQIVV------EY SEQ ID NO: 81 DGGVDKYK-NYIDTIROIVW------EY SEQ ID NO: 82 DGGVDKYK-NYIDTIROIVW------EY SEQ ID NO: 83 DGGVAKYK-NYIDTIROIVV------EY SEO ID NO: 84 DGGVOHYK-DYIDSIREILV------EY SEO ID NO: 85 DNGVEHYK-SYIDSIREIILV------OY SEO ID NO: 86 DGGVEKYK-AYIDSIREQVE------KY SEQ ID NO: 87 DGGVEKYK-AYIDSIREQVE------TY SEQ ID NO: 88 NNGVANYK-AYIDSIVAQLK------AY SEQ ID NO: 89 NNGVALYK-QYIDSIREQLT------TY SEQ ID NO: 90 KDGKNKYK-AYIAD-QGILQ------DY SEQ ID NO: 91 KDGANKYK-AYIAKIKGILQ------NY SEQ ID NO: 92 NGGVANYK-TYNARKILL------EY SEQ ID NO: 93 DGGVEKYK-EYDARAHAW------EY SEQ ID NO: 94 DGGVEKYK-AYIDSIREQVE------Ty SEQ ID NO: 95 DGGVVKYK-AYIDAIRKOLL------AY SEQ ID NO: 96 NNGANNYK-RYIDRIRELII ------OY SEQ ID NO: 97 NNGANNLO-RYIDRIRELLI------OY SEO ID NO: 98 DGGVAKYK-NYIDEIRKLVI------KY SEO ID NO: 99 NNGANNYK-GYNRREIIL------SF SEQ ID NO: 100 NINGVINNYK-AYINRIREIILI------SF SEQ ID NO: 101 EGGVEKYKTQYIDKIAAIIK------KY SEQ ID NO: 102 NDGLNKYK-NYVDQIAAQIK------QF SEQ ID NO: 103 DGGQAKYY-DYIDQIVAQIK------KE SEQ ID NO: 104 DNGEANYH-DYIDQIVAQIK------OY SEQ ID NO: 105 DNGEANYH-DYIDQIVAQIK------OY SEQ ID NO: 106 DGGEEKYR-GYIDGIREQIE------KY SEQ ID NO: 107 NNGQANYE-NYIDQIVAQIQ------QF SEQ ID NO: 108 NNGLNNYK-GYIDQLVAQIK------KY SEQ ID NO : 109 --ELDRYKTEYIDKIAEIILK------AH SEQ ID NO: 110 NNGLANYK-NYVDQIVAOIARTCCPLVTSAITDLACLSEY SEQ ID NO: 111 DAGAENYK-DYIDQIVPOIK------QF SEQ ID NO: 112 --ELSRYKTEYIDAIVKILK------AH SEQ ID NO: 113 --ELAKYKSQYIDPIAALLK------KY

Figure 9D U.S. Patent Jan. 19, 2016 Sheet 25 Of 31 US 9.238,806 B2

SEQ ID NO: 3 210 SDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYA 249 SEQ ID NO: 79 SDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYA SEQ ID NO: 80 SDIRTILL VIEPDSLANLVTNLGTPKCANAPSAYLECINYA SEQ ID NO: 81 SDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYA SEQ ID NO: 82 SDIRTLLVIEPDSLANLVTNLGTPKCANAQSAYLECINYA SEO ID NO: 83 SDIRTILVIEPDSLANLVTNLGTPKCANAOSAYLECINYA SEQ ID NO: 84 SDVHVILVIEPDSLANLVTNLNVAKCANAQSAYLECTNYA SEO ID NO: 85 SDVHTILL VIEPDSLANLVTNLNVAKCANAESAYLECTNYA SEQ ID NO: 86 SDTQIILVIEPDSLANLVTNLNVOKCANAODAYLECTNYA SEQ ID NO: 87 SDVOTILIIEPDSLANLVTNLDVAKCANAESAYLECTNYA SEQ ID NO: 88 PDVHTILIIEPDSLANMVTNLSTAKCAEAOSAYYECVNYA SEQ ID NO: 89 SDVHTILVIEPDSLANVVTNLNVPKCANAQDAYLECINYA SEQ ID NO: 90 SDTRIILVIEPDSLANMVTNMNVPKCANAASAYKELTIHA SEQ ID NO: 91 SDTKVILVIEPDSILANLVTNLNVDKCAKAESAYKELTVYA SEQ ID NO: 92 SDIRTILVIEPDSLANLVTNTNVAKCANAASAYRECTNYA SEQ ID NO: 93 SDTNIILIIEPDSLANLVTNLNVEKCANAQDAYLECTNYA SEO ID NO : 94 SDVOTILIIEPDSLANLVTNLDVAKCANAOSAYLECTNYA SEQ ID NO: 95 SDVRTILVIEPDSLANMVTNMGVPKCAGAKDAYLECTIYA SEO ID NO: 96 SDIRTILVIEPDSLANMVTNMNVOKCSNAASTYKELTVYA SEQ ID NO: 97 SDIRTILVIEPDSLANMVTNMNVOKCSNAASTYKELTVYA SEO ID NO: 98 NDLRIILVIEPDSLANMVTNMNVAKCONAASAYRECTNYA SEQ ID NO: 99 SDWRTILVIEPDSLANMVTNMNVAKCSGAASTYRELTIYA SEQ ID NO : 100 SDVRTILVIEPDSILANMVTNMNVPKCSGAASTYRELTIYA SEQ ID NO: 101 PDVKINLAIEPDSLANMVTNMGVOKCSRAAPYYKELTAYA SEQ ID NO : 102 PDVSVVAVIEPDSLANLVTNLNVOKCANAQSAYKEGVIYA SEQ ID NO: 103 PDVRVIAVIEPDSLANLVTNLNVOKCANAOTTYKACVTYA SEQ ID NO : 104 PDVHVVAVIEPDSLANLVTNLSVAKCANAQTTYLECVTYA SEQ ID NO : 105 PDVHVVAVIEPDSLANLVTNLSVAKCANAQTTYLECVTYA SEQ ID NO : 106 PDVRVALVIEPDSLANMVTNLNVPKCAESEQAYRDGVAYA SEQ ID NO: 107 PDVRVVAVIEPDSLANLVTNLNVQKCANAKTTYLACVNYA SEQ ID NO: 108 PDVRVVAVIEPDSLANLVTNLNVSKCANAQTAYKAGVTYA SEQ ID NO: 109 SNTAFALVIEPDSLPNLVTNSDLQTCQQSASGYREGVAYA SEQ ID NO: 110 POIRVVAVVEPDSLANMVTNLNVPKCAGAOAAYTEGVTYA SEO ID NO: 111 PDVRVVAVIEPDSLANLVTNLNVOKCANGG-TYKASVTYA SEQ ID NO: 112 PKTAFALVIEPDSLPNLVTNSDLOTCKDSASGYRDGVAYA SEQ ID NO : 113 NNHAFALLIEPDSLPNLVTNSDLSACQOSAAGYRDGVAYA

Figure 9E

U.S. Patent Jan. 19, 2016 Sheet 27 Of 31 US 9.238,806 B2

SEQ ID NO:3 290 ASSPRALRGLATNVANYNGWNIT.----SPPSYTQGNAVYN 325 SEQ ID NO: 79 ASSPRALRGLATNVANYNGWNIT----SPPSYTQGNAVYN SEQ ID NO: 80 ASSPRALRGLATNVANYNGWNIT----SPPSYTQGNAVYN SEQ ID NO: 81 ASSPRALRGLATNVANYNGWNIT----SPPSYTQGNAVYN SEQ ID NO: 82 ASSPRALRGLATNVANYNGWNIT----SPPSYTQGNAVYN SEQ ID NO: 83 ASSPSALRGLATNVANYNGWNIT----SPPSYTQGNAVYN SEQ ID NO: 84 AGSPAALRGLATNVANYNAWAID----TCPSYTQGNSVCD SEQ ID NO: 85 ASSPAAVRGLATNVANYNAWTIS----SCPSYTQGNSVCD SEQ ID NO: 86 ASSPAAVRGLATNVANYNAFSID----SCPSYTQGSTVCD SEQ ID NO: 87 ASSPAAVRGILATNVANFNAWSID- - - - SCPSYTSGNDVCD SEQ ID NO: 88 ASAPASLRGATNVANYNAWSS- - - - SPPSYTSGDSNYD SEQ ID NO: 89 ASSPASVRGLATNVANYNAWSIS----RCPSYTQGDANCD SEO ID NO: 90 AGKPSRRGILWTNWSNYNAWKS - - - - SKPDYTESNPNYD SEO ID NO: 91 AGKPSRVRGILWTNWSNYNGWKLS----TKPDYTESNPNYD SEQ ID NO: 92 AGKPKSWRGILWTNWSNYNGWSLS-- - - SAPSYTTPNPNYD SEQ ID NO: 93 AGAPAALRGLATNVANYNAFSID----TCPSYTSONAVCD SEQ ID NO: 94 ASSPAAVRGIATBWANFNAWSID- - - -TCPSYTSGNDVCD SEQ ID NO: 95 AGKPSQLRGMATNVANYNAWDLT----TAPSYTTPNPNFD SEQ ID NO: 96 AGRPAAWRGILATNVANYNAWSIA- - - - SPPSYTSPNPNYD SEQ ID NO: 97 AGRPAAVRGILATNWANYNAWSIA- - - - SPPSYTSPNPNYD SEQ ID NO: 98 AGSPKSWRGILAINVSNYNAWSWS - - - - SPPPYTSPNPNYD SEQ ID NO: 99 AGKPRAVRGILATNVANYNAWSIS----SPPPYTSPNPNYD SEQ ID NO : 100 AGKPRAVRGILATNVANYNAWSVS.----SPPPYTSPNPNYD SEQ ID NO : 101 AGSPRGVRGIVTNVSNYNALRVS----SCPSITQGNKNCD SEQ ID NO : 102 AGSPRNLRGIATNVANFNALRAS ----SPDPITQGNSNYD SEO ID NO : 103 ANSSKFVRGLATNVANYNALSAA- - - - SPDPITOGDPNYD SEO ID NO : 104 AGSPSGVRGLATNVANYNALVAT----TPDPITOGDPNYD SEO ID NO : 105 AGSPSGVRGLATNVANYNALVAT----TPDPITOGDPNYD SEQ ID NO: 106 AGRPKSTRGFATNWSNYNGYSLS----TAPPYTEPNPNFD SEQ ID NO: 107 AGKSPFIKGLATNVANYNALOAA- - - - SPDPITQGNPNYD SEQ ID NO: 108 AGSPOYVRGLATNVANYNALSAS----SPDPWTQGNPNYD SEQ ID NO: 109 AGSPSQVRGISTNVAGWNAWDQEPGEFSDASDAQYNKCON SEQ ID NO:110 AGSPSFFRGILATNWANYNLLNAP- - - - SPDPWTSPNANYD SEQ ID NO: 111 ADFVAFVRAFATNVREYNALTAA----FPRPITQGNPNYD SEQ ID NO: 112 AGSPKQVRGIATNVAGWNQWDLTPGEFSKASDAKYNKCON SEQ ID NO: 113 AGSPKQFRGFATNVAGWNAWDLTPGEFSSASDAQWNKCON

Figure 9G U.S. Patent Jan. 19, 2016 Sheet 28 of 31 US 9.238,806 B2

SEQ ID NO:3 326 EKLYIH------AIGPLLANHGWSNAFFI 348 SEQ ID NO: 79 EKLYIH------AGRILLANHGWSNAFF SEO ID NO: 80 EKLYIH------AGPLLANHGWSNAFE SEQ ID NO: 81 EQLYIH------AGPLLANHGWSNAFF SEQ ID NO: 82 EQLYIH------AGPLANHGWSNAFF SEQ ID NO: 83 EKLYIH------AGPLLANHGWSNAFFI SEO ID NO: 84 EKDYIN------ALAPLLRAOGFD-AHFI SEQ ID NO: 85 EQQYIN------AIAPLLQAQGFD-AHFI SEQ ID NO: 86 EKTYIN------NFAPOLKSAGFD-AHFI SEQ ID NO: 87 EKSYIN------AAPESSAGED-AHE SEQ ID NO: 88 EKLYIN------ALSPLLTSNGWPNAHF SEO ID NO: 89 EEDYWN------ALGPLFOEOGFP-AYFI SEQ ID NO: 90 EQKYIH------ALSPLLEQEGWPGAKFI SEQ ID NO: 91 EQRYIN------AFAPLLAQEGWSNVKFI SEQ ID NO: 92 EKKYIE------AFSPLLNAAGFP-AQFI SEO ID NO: 93 EKGYIN------SFAPELSAAGWD-AHE SEQ ID NO: 94 EKSYIN------AFAPELSXAGFD-AHF SEQ ID NO: 95 EKKYIS------AFAPLAAKGWS-AHF SEQ ID NO: 96 EKHYIE------AFAPLLRNQGFD-AKFI SEQ ID NO: 97 EKHYIE------AFAPLLRNOGFD-AKFI SEQ ID NO: 98 ERHFVE------AFAPLLRQNGWD-AKFI SEQ ID NO: 99 EKHYIE------AFRPLLEARGFP-AQFI SEQ ID NO: 100 EKHYIE------AFRPLLEARGFP-AQFI SEQ ID NO: 101 EERYIN------AAPLLKNEGEP-AHF SEO ID NO: 102 EIHYI------EALAPMLSNAGEP-AHE SEQ ID NO: 103 EILHYIN------ALGPMLAQQGFP-AOFV SEQ ID NO: 104 EMILYIE------AAPLLGS--FP-AHF SEQ ID NO: 105 EMILYIE------AAPLLGS--EP-AHE SEO ID NO: 106 EVRYIN------AFRPLLEARGEP-AYEI SEQ ID NO: 107 EIHYIN------ALAPLLOOAGWD-ATFI SEQ ID NO: 108 EILHYIN------ALAPALQSGGFP-AHFI SEQ ID NO: 109 EKIYIN------TEGAELKSAGMP-NHA SEQ ID NO: 110 EHYINVSDCEVLIWSLTICALAPESSRGFP-AHE SEO ID NO: 111 EFPYIQ------RVRPMLKSPGFP-AOFV SEQ ID NO: 112 EKLYLD------NEGPALKSAGMP-NHA SEQ ID NO: 113 EKYVE------TEGPLLKNAGMP-NHA

Figure 9H

U.S. Patent Jan. 19, 2016 Sheet 31 of 31 US 9.238,806 B2

SEQ ID NO : 3 427 AG ------AWFOAYFVOLLTNANPSFL- 447 SEQ ID NO : 79 AG------AWFQAYFVQLLTNANPSFL SEO ID NO : 80 AG------AWFOAYFVOLLTNANPSFL SEQ ID NO : 81 AG------AWFQAYFVOLLTNANPSFL SEQ ID NO : 82 AG------AWFOAYFVOLLTNANPSFL SEQ ID NO: 83 AG------AWFOAYFVOLLTNANPSFL SEQ ID NO : 84 AG------TWFQAYFVOLLONANPSF-- SEQ ID NO : 85 AG------TWFOAYFVOLLTNANPAF-- SEQ ID NO : 86 AG------TWFQAYFEQLVENANPSL-- SEO ID NO : 87 AG------TWFOAYFEQLLTNANPSL.-- SEQ ID NO : 88 AG------TWFQAYFVOLLTNANPALV SEO ID NO : 89 AG------TWFOAYFEOLLTNANPLF-- SEQ ID NO : 90 AG------TWFOAYFEQLLKNANPSFL SEQ ID NO : 91. AG------TWFQAYFEQLLDNANPSFL SEQ ID NO : 92 AG------OWFOAYFEQLLKNANPAF-- SEQ ID NO : 93 AG------TWFQAYFEQLVANANPPLSS SEQ ID NO : 94 AG------TWFOAYFEQLLTNANPSL.-- SEQ ID NO : 95 AG------QWFOKYFEMLLTNANPPL.-- SEO ID NO : 96 AG------QWFOAYFEOLLINANPPL.-- SEQ ID NO : 97 AG------QWFQAYFEQLLINANPPF-- SEQ ID NO : 98 AG------EWFOAYFEQLLRNANPAF-- SEQ ID NO: 99 AG------QWFOAYFEQLLRNANPPF-- SEQ ID NO : 1 OO AG------QWFNEYFIQLLRNANPPF-- SEQ ID NO : 101 AG------TWFOAYFEMLLKNANPALA SEQ ID NO : 1 O2 AG------TWFQAYFETLVANANPAL-- SEQ ID NO : 103 AG------TWFOTYFETLVSKANPPL.-- SEQ ID NO : 104 AG------TWFQAYFETLVEKANPPL-- SEO ID NO : 105 AG------TWFOAYFETLVEKANPPL.-- SEQ ID NO : 106 AG------QWFOAYFEQLLRNANPPF-- SEQ ID NO : 107 AG------TWFOAYFOTLVSAANPPL.-- SEQ ID NO: 108 AG------QWFOAYFETLVRNASPPL.-- SEQ ID NO : 109 AG------TWNQAYFEMLLKNANPSF-- SEQ ID NO : 110 AMASVYSHSSFOAYFEMLVANAVPAL-- SEQ ID NO : 111 AG------TWFQAYFETLVSKPTRPL-- SEO ID NO : 112 AG------SWNODYFEMLVKNAKPSF-- SEQ ID NO : 113 AG------QWNQDYFEMLLRNAKPQF--

Figure 9K US 9.238,806 B2 1. 2 CELLULASE ENZYME MIXTURES FOR 26, 44, 45, 48, 51, 61 and 74. Further, cellulase in some of the DEPILLING AND USES THEREOF larger GH Families may be grouped into subfamilies. Under the GH system, all cellulase catalytic domains, CROSS-REFERENCE TO RELATED whether CBH or EG enzymes, are designated by “Cel' fol APPLICATIONS lowed by the GH Family number. For example, cellulase comprising catalytic domains belonging to GH Families, 5, 6 This application is a 35 U.S.C. 371 national application of and 7 may be referred to as Cel5, Celé and Cel7 cellulases, PCT/CA2012/050074/filed Feb. 9, 2012, which claims prior cellobiohydrolases or endoglucanases or as Family 5, Family ity or the benefit under 35 U.S.C. 119 of U.S. provisional 6, and Family 7 cellulases, cellobiohydrolases or endogluca 10 nases. Designations such as Cel5, Cel6 and Cel7 may be application No. 61/440,985 filed Feb. 9, 2011, the contents of followed by the capital letters A, B, C and so forth, which which are fully incorporated herein by reference. indicate the order in which the enzymes of the same family were identified from the source organisms. Further, the Cel FIELD OF THE INVENTION designations may be preceded by a genus, species identifier 15 such as “Tr” for Trichoderma reesei. The present invention relates to a cellulase enzyme mixture Fungi such as Trichoderma secrete a number of different and uses thereof, in particular to a cellulase enzyme mixture cellulase enzymes (also referred to herein as an “enzyme for use in treating cellulose-containing goods. mixture”, “cellulase mixture' or “cellulase enzyme mixture') that are individually known as components. The more preva BACKGROUND OF THE INVENTION lent of these enzyme components include cellobiohydrolase (CBH), also called exo-1,4-beta-D-glucanases, endogluca Cellulase enzymes are widely used to improve the appear nase (EG), or endo-1,4-beta-D-glucanases, and beta-glucosi ance and softness of cellulose-containing fabrics. A wide dase enzymes. This classification is based on the enzymes spread application of cellulase enzymes is to remove cotton Substrate specificities, i.e. affinity towards the chain ends fuZZ and loose surface fibers in or on the fabric. This process 25 (exo), the interior of the glucose chain (endo), or glycosidic is referred to as “depilling”, “biopolishing and “biofinish bonds of soluble cellooligosaccharides and cellobiose (beta ing and smoothes the surface of the fabric, which in turn glucosidase). improves its softness and appearance. Cellulase treatment Trichoderma reesei is a widely studied and industrially also aids in the prevention of subsequent formation of fiber important fungus for the production of cellulases. It produces pills that make the garments appear worn. During depilling it 30 at least six genetically different cellulases: two cellobiohy is desirable to minimize strength loss of the fabric due to the drolases, referred to as Cel6A (SEQ ID NO:3) and Cel7A hydrolytic action of the cellulases. (SEQID NO:5), also known as CBHII and CBHI, and at least Another industrial application of cellulase enzymes is for four endoglucanases Cel5A (SEQID NO:2), Cel7B (SEQID treating denim fabrics So as to impart to them a “stone NO:6), Cel12A and Cel45A (SEQ ID NO:4), also known as washed' appearance. Such a process is known in the industry 35 EGII, EGI, EGIII and EGV. respectively. Some of these cel as “bio-stoning. The term bio-stoning was adopted as pum lulase components have been identified as contributing to ice stones were traditionally used to treat the fabric. However, improvements in depilling, while others are seen as detrimen cellulases have largely replaced pumice stones in recent tal as they can cause fabric strength loss, as discussed below. years. Bio-stoning is quite different from depilling: bioston Efforts have been made to improve the properties of cellu ing aims to remove colour from denim and control its re 40 lase mixtures for textile depilling by increasing the propor deposition on the fabric while depilling aims to solely tion of endoglucanase components in the secreted enzyme improve softness and appearance as in depilling. mixture relative to the natural mixture. For instance, WO Cellulase enzymes are a group of glycoside hydrolase 92/17574 discloses an approach that involves adjusting the enzymes that catalyze the hydrolysis of beta-1,4-glycosidic amounts of EG type components relative to CBH I type linkages in the cellulose polymer and often comprise a cellu 45 components (CelTA) so that the protein weight ratio of the EG lose binding domain (CBD) and a catalytic domain. A region to CBH I type components is greater than 5:1. Cotton-con between these two domains known as a “linker” or “linker taining fabrics treated with Such compositions exhibited peptide' serves as a flexible spacer between the CBD and the decreased strength loss during depilling treatment compared catalytic domain. The catalytic domains of individual cellu to fabrics containing greater amounts of CBH I type (CelTA) lase components are classified by both the Enzyme Commis 50 components. In addition, EP866 165 discloses enzyme com sion (EC) and the Glycoside Hydrolase (GH) family systems. positions enriched in EG II (Cel5A), which exhibit improve The Enzyme Commission distinguishes two classes of cellu ments in depilling efficiency and reduced strength loss. In lases based on their preference for cleavage of internal beta-1, embodiments, the enzyme compositions contain a cellulase 4 linkages (endoglucanase or “EG', EC 3.2.1.4) or the release protein content of at least 95% EG II. of cellobiose from the reducing or non-reducing end of the 55 U.S. Pat. No. 6,162,782 discloses that reduced strength cellulose polymer (cellobiohydrolases or “CBH', EC 3.2.1. loss of cotton fabrics can be achieved with cellulase detergent 91, Sometimes also referred to as exoglucanases). In contrast, compositions that contain one or more endoglucanase com the GH family system distinguishes the catalytic domains of ponents and reduced levels (less than 5 wt %) of CBH I cellulase components based on the conservation of primary (CelTA). In these studies, it was found that CBH I, and to a and secondary structure, as well as the stereochemistry of the 60 certain extent CBH II, played a role in strength loss. The catalytic reaction. The GH family designations for all known EG-enriched detergent compositions were reportedto impart cellulase catalytic and binding domains is provided and con improvements in Softening, washing and color restoration. tinually updated through the Carbohydrate-Active Enzymes Similarly, Miettinen-Oinonen et al. (Applied and Environ (CAZy) database (Cantarel et al., 2009, Nucleic Acids Res mental Microbiology, 2002, 68(8):3956-3964) reported 37:D233-238) available at the URL: cazy.org. Cellulase 65 improvements in visual appearance and reductions in pilling enzymes may be found in a number of GH Families includ tendency with EG II-enriched cellulase preparations devoid ing, but not limited to, Families 5, 6,7,8,9, 10, 12, 16, 18, 19. of CBH I and CBH II. Miettinen-Oinonen et al. (VTT Publi US 9.238,806 B2 3 4 cations 550, 2004, Trichoderma reesei strains for production enzymes in biostoning, alone or in combination with other of cellulases for the textile industry) also report good depill cellulase components, including endoglucanases. However, ing results with EG II-enriched cellulase preparations. The the depilling properties of these enzyme compositions were use of endoglucanases in textile applications is also discussed not tested in these studies. in WO 2004/053039, EP 495 258 and U.S. Pat. Nos. 6,001, 5 Synergy between cellulase components has been reported 639, 5,958,082 and 5,948,672. in the literature in the hydrolysis of cellulosic substrates, or Moreover, a number of groups have contemplated the use complete degradation and conversion of cellulose into ulti of Cel45 cellulases in depilling. WO 97/14804 discloses a mately glucose (Gusakov et al., Biotechnology and Bioengi neutral Cel45A cellulase (20 K cellulase) from Melanocar neering, 2007,97(5):1028-1038; Andersenetal, Enzyme and pus origin for use in the textile and detergent industry . . . . 10 Microbial Technology, 2008, 42(4):362-370; Boisset al., Bio WO2010/076388 discloses the production and use of Geo technology and Bioengineering, 2001, 72(3):339-345; Iga myces or Fusarium Cel45 endoglucanases in denim washing rashi et al., Applied and Environmental Microbiology, 2008, and depilling. U.S. Publication No. 2007/011 1278 discloses 74:5628-5634; and Zhanget al., Biotechnology and Bioengi the use of STCE1. a Cel45 endoglucanase, derived from neering, 2004, 7(88):797-824). However, extended hydroly Staphylotrichum, in washing or depilling of cellulose-con 15 sis is generally not a desired effect in depilling, as it may taining fabrics. U.S. Pat. No. 7,741,093 discloses fusion of result in destruction of the fabric or severe strength loss. the Melanocarpus Cel45 endoglucanase to a linker peptide of Furthermore, as reported by Ramos et al. (Biocatalysis and Trichoderma reesei CBH I and a cellulose binding domain for Biotransformation, 2007, 25(1):35-42) hydrolysis may not bio-stoning and biofinishing. The purpose of constructing correlate with biopolishing effects. Such fusion proteins was to increase the size of the Melano Despite these efforts, there is still a need for improved carpus Cel45A enzyme, thereby decreasing the ability of the combinations of cellulase enzymes and compositions thereof enzyme to penetrate the fabric, which in turn reduces strength that are more effective in depilling of cellulose-containing loss. Similar approaches with Cel45 endoglucansaes and goods. In particular, there is a continuous need for more other cellulase enzymes are disclosed in WO 2007/118935 efficient cellulase enzyme mixtures to improve the process and U.S. Pat. No. 7,256,032. 25 economics. The present invention aims to meet these needs. Other groups have focused on elucidating whether or not cellulase enzyme components synergize with one another. SUMMARY OF THE INVENTION The identification of synergistic combinations of enzyme components that provide for enhanced depilling could be a It is an object of the present invention to provide an step forward with respect to improving process economics. 30 improved cellulase enzyme mixture for use in treating cellu Such improvements may be achieved since less enzyme pro lose-containing goods. tein, which is costly, would be necessary to impart the desired According to a second aspect of the invention, there is depilling effect. provided a depilling composition comprising an enzyme mix Heikinheimo and Buchert (Textile Research Journal, 2001, ture, which enzyme mixture comprises a Family 45 cellulase 71 (8):672-677) investigated the depilling properties of Tri 35 enzyme component and one or more additional cellulase choderma reesei EG I and II and CBH I and II cellulase enzyme components selected from a Family 5 cellulase, a components alone and in combination. Treatment of cotton Family 6 cellulase or a combination thereof. The enzyme interlock fabric with EG II-based combinations with CBHI or mixture is secreted by a genetically modified microbe over CBH II resulted in favourable depilling properties. However, expressing (i) a Family 45 cellulase gene encoding said Fam the investigators also reported decreased depilling activity for 40 ily 45 cellulase enzyme; and (ii) a gene or genes encoding the combinations of the two endoglucanases, EG I and EG II. one or more additional cellulase enzyme component selected That is, no endo-endo Synergy between the cellulase compo from a Family 5 cellulase, a Family 6 cellulase or a combi nents was observed. nation thereof, wherein the Family 45 cellulase has at least Cavaco-Paulo and Almeida (Textile Chemist and Colorist, 75% sequence identity to amino acids 1-213 of SEQID NO:7 1996, 28(6):28-32) observed a high activity of EGI and II 45 (HiCel45) or at least 20% sequence identity to amino acids deleted Trichoderma reesei cellulase mixtures on cotton cel 1-166 of SEQID NO.4 (TrCel45). lulose. The authors state therein that the effect may possibly According to one embodiment of the invention, the depill be due to synergy between the two CBH components or the ing composition comprises both the Family 45 cellulase and CBH components and residual EG III or EGV. In Cavaco the Family 5 cellulase. In a further embodiment of the inven Paulo, Carbohydrate Polymers, 1998, 37:273-277, it was 50 tion, the depilling composition further comprises the Family stated that minor EG components seem to cooperate with the 6 cellulase. In yet another embodiment of the invention, the CBHs, in a synergistic fashion, to fully hydrolyse cotton. depilling composition further comprises a Family 7 cellulase. However, no testing was carried out to examine which par Alternatively, the depilling composition lacks a Family 7 ticular components exhibited synergism with one another. cellulase. U.S. Pat. No. 5,958,083 discloses binary cellulase enzyme 55 The Family 5 cellulase may comprise a substitution of an mixtures for use in bio-stoning. The first component is a amino acid at position363 with a non-native alanine, serine or Family 5 endoglucanase derived from Bacillus or threonine, which position is determined from alignment of Clostridium, or Family 7 endoglucanase derived from Humi the modified Family 5 cellulase with a wild-type Trichoderma collainsolens. The second component is a mechanical abrad reesei Cel5A amino acid sequence as set forth in SEQ ID ing agent, and/or an abrading cellulase (to form localized 60 NO:2. variation in color density), which may be a Family 12 or a The present invention also provides a process for depilling Family 45 cellulase with a cellulose binding domain. that comprises a step of contacting cellulose-containing Although improved bio-stoning with low backstaining was goods with the depilling composition set forth above. obtained, the properties of these compositions in depilling According to a further aspect of the invention, there is assays were not investigated. 65 provided a genetically modified microbe overexpressing (i) a Miettinen-Oinonen et al. (Enzyme and Microbial Technol Family 45 cellulase gene encoding said Family 45 cellulase ogy, 2004, 34:332-341) examined the effect of Family 45 enzyme; and (ii) a gene or genes encoding the one or more US 9.238,806 B2 5 6 additional cellulase enzyme component selected from a Fam derma reesei Family 5 cellulase abbreviated TrCel5A (SEQ ily 5 cellulase, a Family 6 cellulase or a combination thereof, ID NO:2). The catalytic amino acids at the equivalent position wherein the Family 45 cellulase has at least 75% sequence 218 and 329 in TrCel5A are indicated with an arrow. The identity to amino acids 1-213 of SEQID NO:7 (HiCel45) or conserved amino acids at the equivalent position 130, 174, at least 20% sequence identity to amino acids 1-166 of SEQ 5 217, 288, and 290 in TrCel5A are indicated with an asterisk. ID NO:4 (TrCel45). For cellulases with a cellulose-binding domain, only the cata According to one embodiment of the invention, the geneti lytic core sequences are presented. cally modified microbe overexpresses at least the Family 45 FIG. 4 is a map of the Trichodermareesei transformation cellulase and the Family 5 cellulase. Alternatively, the geneti vector pP7a-TrCel45A-hph-TV. cally modified microbe overexpresses the Family 6 cellulase. 10 FIG. 5 is a map of the Trichodermareesei transformation According to a further embodiment of the invention, the vector pP7a-xyn2ss-Cel45A-hph-TV. genetically modified microbe expresses a Family 7 cellulase. FIG. 6 is a map of the Trichodermareesei transformation Alternatively, the genetically modified microbe does not vector pP6a-6Ass-Cel45A-ble-TV. express a Family 7 cellulase. FIG. 7 depicts the relative abundance of TrCel7A, The genetically modified microbemay express a Family 5 15 TrCeléA, TrCel45A and TrCel5A in cellulase mixtures pro cellulase that comprises a Substitution of an amino acid at duced by Trichoderma reesei transformants overexpressing position 363 with a non-native alanine, serine or threonine, TrCel45A and a modified TrCel5A, and by the parental strain which position is determined from alignment of the modified P998A as assessed in a final fermentation sample by ELISA Family 5 cellulase with a wild-type Trichoderma reesei using component specific antibodies. The strain names are Cel5A amino acid sequence as set forth in SEQID NO:2. indicated under each bar and the relative abundance of each The Family 45 cellulase and the one or more additional cellulase tested is indicated on the corresponding bars. cellulase enzyme component selected from a Family 5 cellu FIG. 8 shows the relative depilling activity of cellulase lase, a Family 6 cellulase or a combination thereof, may be enzyme mixtures produced by Trichoderma reesei transfor expressed from coding sequences in said genetically modi mants overexpressing TrCel45A and a modified TrCel5A, fied microbe that are endogenous or heterologous to the 25 and by parental strains BTR213 and P998A. The strain names microbe. are indicated under each bar and the relative depilling activity According to another aspect of the invention, there is pro is indicated on the corresponding bar. vided a depilling composition comprising an enzyme mix FIGS.9A,9B,9C,9D,9E,9F,9G,9H,9I,9J, and 9K show ture, which enzyme mixture comprises (i) a Family 45 cellu an amino acid sequence alignment of 35 selected Glycoside lase enzyme component; and (ii) at least one or more 30 hydrolase (GH) Family 6 cellulases to amino acids 83-477 of additional cellulase enzyme components that are selected a Trichoderma reesei Family 6 cellulase abbreviated TrCeléA from the group consisting of a Family 5 cellulase, a Family 6 (SEQID NO:3). The Family 6 cellulases correspond to those cellulase, and a combination thereof, wherein said enzyme provided in Table 4 and appear in the order presented in the mixture is characterized by having a weight ratio of the Fam table. ily 45 and the Family 5 cellulase enzyme components or the 35 FIG. 10 is a map of the Trichoderma reesei transformation Family 45 and the Family 6 cellulase enzyme components vector pChHiCel5A-pyra-TV. that exhibits synergy in an assay that measures specific depill FIG. 11 is a map of the Trichoderma reesei transformation ing activity. vector PC/X-TriCel45a-ble-TV. Disclosed herein are combinations of cellulase enzymes FIG. 12 is a Western blot of culture filtrates of Trichoderina that are particularly effective in the depilling of cotton-con 40 reesei transformants of parental strain P579A expressing taining goods. The Applicants have identified combinations HiCel45A (P1622A and P1622F). of cellulase enzyme components that provide for enhanced FIG. 13 shows the relative depilling activity of cellulase depilling of cotton-containing goods relative to the combined enzyme mixtures produced by Trichoderma reesei transfor depilling effect of the individual enzyme components. The mants overexpressing HiCMC3 (strain P579A) or overex utilization of Such enzyme combinations could be a step 45 pressing both HiCMC3 and HiCel45A (strain P1622A), rela forward with respect to improving process economics. Such tive to that produced by parental strains BTR213. The strain improvements may be achieved since less enzyme protein, names are indicated under each bar and the relative depilling which is costly, would be necessary to imparta desired depill activity is indicated on the corresponding bar. ing effect. FIG. 14 shows the depilling activity (in terms of the per 50 centage of fines released from a fabric sample) by increasing BRIEF DESCRIPTION OF THE DRAWINGS dosages of purified TrCel45A, purified TrCel5A-G363 A, or a blend of TrCel45A-TrCel5A-G363 A. FIG. 1 shows an amino acid sequence alignment of eight FIG. 15 shows the depilling activity (in terms of the per selected Glycoside hydrolase (GH) Family 45 cellulases to centage of fines released from a fabric sample) by increasing amino acids 1-166 of a wild-type Trichoderma reesei Family 55 dosages of purified TrCel45A, a blend of TrCel6A+TrCel5A 45 cellulase abbreviated TrCel45A (SEQ ID NO:4). Con G363 A-TrCel5A or a blend of TrCel45A-TrCel6A+ served amino acid motifs characteristic of Family 45 cellu TrCel5A-G363A-TrCel5A. lases are underlined. FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show an amino acid DETAILED DESCRIPTION OF THE INVENTION sequencealignment of 49 selected Glycoside hydrolase (GH) 60 Family 45 cellulases to amino acids 1-213 a Humicolainso The following description is of a preferred embodiment by lens Family 45 cellulase abbreviated HiCel45A (SEQ ID way of example only and without limitation to the combina NO:7). Conserved amino acid motifs characteristic of Family tion of features necessary for carrying the invention into 45 cellulases are underlined. effect. The headings provided are not meant to be limiting of FIGS. 3A, 3B, 3C, 3D, 3E, 3F, and 3G show an amino acid 65 the various embodiments of the invention. Terms such as sequence alignment among 14 selected Glycoside hydrolase “comprises”, “comprising”, “comprise”, “includes”, “includ (GH) Family 5 cellulases to amino acids 71-397 of a Tricho ing” and “include are not meant to be limiting. In addition, US 9.238,806 B2 7 8 the use of the singular includes the plural, and “or” means sequence of H/Q/D/N-F/L-D-I/L/F; or (ii) has the second “and/or unless otherwise stated. Unless otherwise defined conserved signature sequence of H/Q/D/N-F/L-D-I/L/F but herein, all technical and scientific terms used herein have the lacks said first conserved sequence. In one embodiment, the same meaning as commonly understood by one of ordinary second conserved signature sequence is H-F-D-I. Such con skill in the art. served signature sequences may be found, for example, at Family 45 Cellulases amino acids 6-19 and 115-118 of SEQID NO: Glycoside hydrolases (GHS) are a large group of enzymes Family 45 cellulase enzymes have been divided into at least that cleave glycosidic bonds between individual carbohydrate two subfamilies referred to as “A” and “B” (Igarashi et al., monomers in large polysaccharide molecules. Cellulases Applied and Environmental Microbiology, 2008, 74(18): cleave the beta 1-4 bond between glucose monomers in the 10 5628-5634). According to one embodiment of the invention, cellulose polymer. GH enzymes all share one of two common the Family 45 cellulase enzyme is a subfamily A member. mechanisms, called inverting and retaining, for introducing a According to another embodiment of the invention, the Fam water molecule at a glycosidic bond thus cleaving the ily 45 cellulase enzyme is a subfamily B member. This polysaccharide. includes, but is not limited to, those subfamily A and subfam The GH Family 45 cellulase enzymes (formerly Family K) ily B enzymes listed in the tables below. It should be under act with inversion of anomeric configuration to generate the 15 stood that amino acid numbering of the Cel45A sequences C-D anomer of the oligosaccharide as a product. It has been disclosed herein are based on the sequences of the mature, elucidated that, in the active site, one aspartic acid amino acid secreted protein i.e., the protein without a secretion signal acts as a general acid and another as a general base. peptide and/or leader peptide The three dimensional structure of Family 45 enzymes has Table 1 and Table 2 below provide a representative list of been elucidated (see, for example, the structure of Humico family members from subfamilies “B” and 'A', respectively. lainsolens in Davies et al., 1996, ActaCrystallographica Sec Sequence identity for each amino acid sequence with a ref tion D-Biological Crystallography 52:7-17 Part 1). The erence subfamily A or B family member is also provided in enzymes contain a six-stranded beta-barrel to which a seventh each table. For the subfamily B sequences in Table 1, Strand is appended. The structure contains both parallel and sequence identity was determined by alignment with amino anti-parallel beta-strands. The active center is located in an 25 acids 1-166 of Trichoderma reesei Cel45A (TrCel45A: SEQ open Substrate-binding groove. ID NO:4) and for the subfamily A sequences in Table 2, As used herein, the term “Family 45 cellulase' or “Cel45” sequence identity was determined by alignment with amino means a carbohydrate active cellulase enzyme that contains a acids 1-213 of Humicolainsolens Cel45A (HiCel45A: SEQ glycoside hydrolase Family 45 catalytic domain that is clas ID NO:7). FIGS. 1 and 2 show amino acid sequence align sified under EC 3.2.1.4. The term encompasses a carbohy 30 ments of the Family 45 cellulases provided in Tables 1 and 2. drate active enzyme that hydrolyzes cellulose and cello-oli respectively, below. It should be noted that the letter appear gosaccharides using an inverting mechanism, and has either ing after the abbreviated name does not denote inclusion of of the following two signature sequences in the vicinity of the the Family 45 in any particular subfamily. For instance, the T. catalytic aspartic acid amino acids: (i) both a first conserved reesei Family 45 cellulase belongs to subfamily B but is signature sequence of A/S/T-T-R/N/T-Y/F/T-X-D-X-X-X-X- abbreviated TrCel45A because it is the first Family 45 cellu X-C/A-A/G/S-W/C and a second conserved signature lase isolated from T. reesei. TABLE 1 Family 45 cellulase subfamily B members and sequence identity with amino acids 1-166 of TrCel45A (%). GenBank SEQ Percent identity with Abreviated Accession ID TrCel45Aamino acids 1-166 Organism Name Number NO: of SEQ ID NO:4 Trichoderma reesei TrCel4SA CAA83846.1 4 1OOO Trichoderma viride TvCV AAQ21385.1 8 1OOO Penicilium decumbens PoCel45A ACF33814.1 9 84.O Aspergilius nidians AnAN6786.2 EAAS8604.1 10 49.4 Haiotis discus discus HEG1 ABO266.08.1 11 36.1 Amplillaria crossean ACEG27I ABR926.37.1 12 34.6 Amplillaria crossean ACEG27II ABR926.38.1 13 33.5 Mytilus edulis MeFg CACS9695.1 14 30.3 Phanerochaete chrysosporium PcCel45A BAG683OO1 15 26.2 *For the purpose of this specification, PcCel45A is categorized as a subfamily B member, Igarashi et al. (supra) define a subfamily “C”Family 45 cellulase and indicate that PcCel45A would be a member of this family, although they report that the enzyme exhibits some similarity to that of subfamily B.

TABLE 2 Family 45 cellulase subfamily A members and sequence identity with HiCel45A (%). GenBank Percent identity with Abreviated Accession SEQID HiCel45Aamino acids 1-213 Organism Name Number NO: of SEQ ID NO: 7 Humicoia insolens HiCel45A. AAE16508.1 7 1OOO Humicola grisea var. HgEgl3 BAA74956.1 16 98.5 thermoidea Humicola nigrescens HnCel45A CAB42308.1 17 85.4 US 9.238,806 B2 10 TABLE 2-continued Family 45 cellulase subfamily A members and sequence identity with HiCel45A (%). GenBank Percent identity with Abreviated Accession SEQID HiCel45Aamino acids 1-213 Organism Name Number NO: of SEQID NO: 7 Staphylo trichumcoccos ScSTCE1 BAG691.87. 18 80.7 porum Melanocarpus albomyces MaCel45A CAD56665. 19 77.9 Podospora aniserina PaCel45A CAP61565. 2O 74.1 Acremonium thermophilum AtSEQ6 ACE10216. 21 73.7 Thielavia terrestris TtCel4SA CAHO31.87. 22 73.7 Trichothecium roseum TroCel45A CAB42312. 23 73.3 Acremonium thermophilum AtSEQ2 ABW41463.1 24 73.2 Fusarium angioides FaCel45A CAB4231O. 25 72.8 Clonostachys rosea f. CrCel45A CAB42311. 26 72.3 catenulata Neurospora crassa NcCel45A CAD70529. 27 69.4 Voluteia coiletotrichoides VcSEQ22 AAYOO854. 28 64.4 Gibbereia zeae GZCel45A AARO2399. 29 62.6 Fusarium oxysportin FoCel45A AAA65589. 30 62.1 Acremonium sp. AsSEQ10 AAYOO848. 31 60.8 Acremonium sp. AsSEQ8 AAYOO847. 32 59.3 Chrysosporium lucknowense CICel45A AAQ38150. 33 57.3 Thielavia heteroihaica ThSEQ2 AAYOO844. 34 57.3 Mucor circineioides McMce1 BAD958O8. 35 56.8 Reticuliternessperatus RshpCel45A BAA98.037. 36 56.8 hindgut protist Bursaphelenchus xylophilus BxEngl BAD34546. 37 55.9 Botryotinia fuckeiana BfCel45A XP OO1547700.1 38 55.8 Acremonium thermophilum AtSEQ4 ABW41464.1 39 55.3 Scopulariopsis brevicautiis SbEgI Q7M4T4* 40 55.0 Syncephalas trumracemosum SrCBHI ABU491.85.2 41 55.0 Rhizopus oryzae RoRoe1 BACS3956. 42 54.7 Crinipeliss cabella CsSEQ16 AAYOO851 43 53.9 Macrophomina phaseolina MpSEQ14 AAY00850 44 53.9 Podospora aniserina PaCe4SB CAP69443 45 53.9 Rhizopus oryzae RORCe3 BACS3988. 46 53.8 Bursaphelenchus xylophilus BxEng2 BAD34544. 47 S3.6 Bursaphelenchus xylophilus BxEng3 BAD34548. 48 S3.6 Humicola grisea war. HgEgl4 BAA74957. 49 53.4 thermoidea Phycomyces initens PnPce1 BAD778O8. 50 53.2 Rhizopus oryzae RoRoe2 BACS3987.1 51 S2.9 Mastoiernes darwiniensis MdhsFm4 CAD392OO. 52 52.7 hindgut symbiont sp. Magnaporihe grisea MgCel45A XP 3634.02.1 53 51.8 Mastoiernes darwiniensis MdhsFm3 CAD391.99. S4 51.8 hindgut symbiont sp. Mastoiernes darwiniensis MdhsFm1 CAD391.97. 55 51.3 hindgut symbiont sp. Mastoiernes darwiniensis MdhsFm2 CAD39198. 56 51.3 hindgut symbiont sp. Pichia pastoris GS115 PpCel45A CAY71902.1 57 51.3 Piromyces equi PeCel45A CAB92325. 58 48.2 Aprionagermari AgCelI AAN783.26. 59 43.8 Aprionagermari AgCelII AAR22385. 60 43.6 Alternaria alternata AaK1 AAFOSFOO.1 61 41.4 Phaedon cochleariae PcEg CAA76931. 62 40.4 Taiaromyces emersonii TeCel45A CA75963.1 63 37.6 Ustilago maydis UmEgl1 AAB36147. 64 33.6 *Uniprot entry

The sequence identities of the Family 45 cellulases 55 performed a second time using the cropped sequences and reported in Tables 1 and 2 above are determined as follows. again, all amino acids that are found before or after the ref The full sequence of each enzyme (including CBM and signal erence enzyme’s first or last amino acid are cropped from the peptide if present) is aligned with either amino acids 1 to 213 alignment. Percent identity with the reference sequences of the reference sequence HiCel45A (SEQ ID NO:7) or 60 selected amino acids is then calculated. amino acids 1 to 166 of the reference sequence TrCel45A Other sequence alignment algorithms are known to those (SEQ ID NO:4) using ClustalW Multiple Alignment tool of skill in the art, including the BLAST algorithm (BLAST (Thompson et al., Nucleic Acids Research, 1994, 22(22): and BLAST 2.0: Altschul et al., Nuc. Acids Res., 1997, 4673-4680), with default settings, found in the BioEdit soft 25:3389-3402; and Altschul et al., J. Mol. Biol., 1990, 215: ware version 7.0.9.0 (Jun. 27, 2007). All amino acids that are 65 403-410; the algorithm disclosed by Smith and Waterman, found before or after the reference enzyme’s first or last Adv. Appl. Math., 1981, 2:482-489, by the homology align amino acids are cropped from the alignment. The alignment is ment algorithm of Needleman and Wunsch, J. Mol. Biol. US 9.238,806 B2 11 12 1970, 48:443-453, by the search for similarity method of manner, mutations introduced in regions of low sequence Pearson and Lipman, J. Proc Natl Acad Sci., 1988, 85(8): conservation among Family 45 cellulases are expected to 2444-8, by computerized implementations of these algo have a lower probability of reducing or altering the catalytic rithms (GAP, BESTFIT, FASTA, and TFASTA in the Wis activity of the enzyme. consin Genetics Software Package, Genetics Computer It will be understood that the modified Family 45 cellulase Group, 575 Science Dr. Madison, Wis.), or by manual align may be derived from any suitable Family 45 cellulase. That is, ment and visual inspection (see, e.g., Current Protocols in it may be derived from a naturally-occurring or “wild-type' Molecular Biology, John Wiley & Sons, Ausubel et al., eds. Family 45 cellulase or from a Family 45 cellulase that already 1995 supplement). When conducting BLAST alignments and contains other amino acid Substitutions, deletions or inser sequence identity determinations for cellulase enzymes, the 10 tions. By the term “wild-type Family 45 cellulase' it is meant amino acid sequences comprising the catalytic domains and a Family 45 cellulase that does not contain any genetic alter CBDS may be considered separately or as one contiguous ation(s) introduced by molecular biology techniques. Sequence. According to one embodiment, the Family 45 cellulase is a Family 5 Cellulases sub-family B cellulase and has 20% to 100% amino acid 15 The amino acid sequences for over 3,000 naturally occur sequence identity to amino acids 1-166 of SEQ ID NO:4 ring Family 5 cellulases of fungal and bacterial origin have (TrCel45A), or more preferably 30% to 100% amino acid been elucidated. Regions of Family 5 cellulases are well sequence identity to amino acids 1-166 of SEQID NO:4. For conserved in most Family 5 cellulase enzymes and this has example, the Family 45 cellulase may comprise an amino allowed the alignment of parts of the catalytic domains of acid sequence that is 20,30,40, 50, 60, 65,70, 75,80, 85,90, family members. 95 or 100% identical to amino acids 1-166 of SEQID NO:4. Table 3 below includes a representative list of 14 known According to a further embodiment, the Family 45 cellu Family 5 cellulases and FIG.3 shows an amino acid sequence lase is a sub-family A cellulase and has 30% to 100% amino alignment among the cellulases provided in the table. The acid sequence identity to amino acids 1-213 of SEQID NO:7 percent sequence identities of the Family 5 cellulases of Table (HiCel45A), or more preferably 40% to 100% amino acid 25 3 are determined using the procedure described previously for sequence identity to amino acids 1-213 of SEQID NO:7. For the Family 45 cellulases. TABLE 3 Family 5 cellulase family members and sequence identity with TrCel5A catalytic domain (%). Percent identity with GenBank TrCel5Aamino acids Abbreviated Accession SEQ ID 71-397 Organism Name Number NO: of SEQID NO: 2 Trichoderma reesei TrCel5A AAA34213.1 2 1OOO Penicilium janthinelium PEgl2 CAA61740.1 65 62.O Macrophomina phaseolina MpEgl2 AABO3889.1 66 S8.1 Cryptococci is fiavits CfEgl1 AAC60541.1 67 43.2 Aspergilius nidians AnEglA EAA65878.1 68 28.8 Aspergilius kawachii AkCel5A BAB62317.1 69 27.1 Macrophomina phaseolina MpEgl1 AABS1451.1 70 27.8 Voivarielia voivacea WvEG1 AAG59832.1 71 27.6 Thermoascus attrantiacus TaEg1 AAL88714.2 72 27.5 Aspergilius actileatus AaCel1 AACO8587.1 73 26.3 Humicoia insolens HCMC3 CAA53631.1 74 26.6 Orpinomyces joyonii OCelB29 AAB69347.1 75 14.4 Acidothermus cellulolyticus AcCel5A AAA75477.1 76 16.0 Bacilius subtiis BSCel5A ACI15227.1 77 14.6 Bacilius cellulosilyticus BCNK1 AAA22299.1 78 14.5 example, the Family 45 cellulase may comprise an amino 50 As used herein, the term “Family 5 cellulase' or “Cel5A' acid sequence that is 30, 40, 50, 60, 65,70, 75, 80, 85,90, 95 encompasses a carbohydrate active cellulase enzyme that or 100% identical to amino acids 1-213 of SEQID NO:7. contains aglycohydrolase (GH) Family 5 catalytic domain The Family 45 cellulase may be a naturally-occurring or that is classified under EC 3.2.1.4. The term encompasses any carbohydrate active enzyme that exhibits at least hydrolysis wild-type Family 45 cellulase or a modified Family 45 cellu 55 of (1->4)-beta-D-glucosidic linkages using a retaining lase. By “modified Family 45 cellulase', it is meanta Family mechanism, including enzymes with conserved R130, H174, 45 cellulase that contains one or more genetic alteration, E217, E218, H288, Y290 and E329 amino acids (determined introduced by genetic engineering techniques, which do not by alignment with a Trichoderma reesei Family 5 cellulase, significantly compromise the structure and function of the SEQID NO:2). enzyme. Such techniques include, but are not limited to, 60 Enzymes of Family 5 share a common (beta/alpha)-barrel site-directed mutagenesis, cassette mutagenesis, random fold and a catalytic mechanism resulting in a net retention of mutagenesis, including that conducted on isolated DNA or by the anomeric Sugar conformation. Glycoside hydrolase exposing a microorganism to a mutagen, such as UV light, catalysis is driven by two carboxylic acids found on the side synthetic oligonucleotide construction, cloning, Sub-cloning, chain of aspartic acid and/or glutamic acid. These two amino amplification by PCR, in vitro synthesis and other genetic 65 acids are highly conserved among family members. engineering techniques. As would be appreciated by those of The Family 5 cellulase of the invention may be a modified ordinary skill in the art, but without being limiting in any Family 5 cellulase comprising at least a mutation at position US 9.238,806 B2 13 14 363 to an alanine, serine or threonine, for example, Cel5A with the invention include enzyme species from the genera of G363A (SEQ ID NO:1). Generally, the amino acid intro Trichoderma, Hypocrea, Penicillium, Botryotinia, Macro duced at position 363 is “non-native’, meaning that that it phonina, Aspergillus, Orpinomyces, Acidothermus, Pestalo does not naturally occur at the corresponding position in the tiopsis and Xvlella. In one example of the invention, the wild-type Family 5 cellulase sequence from which it is modified Family 5 cellulase is derived from species selected derived. from the group consisting of Trichoderma reesei (SEQ ID By “modified Family 5 cellulase', it is meanta Family 5 NO:2), Trichoderma viride, Hypocreajecorina, Penicillium cellulase that contains one or more genetic alteration intro decumbens, Penicilliumjanthinellum, Botryotiniafuckeliana, duced by genetic engineering techniques that do not signifi Xylellafastidiosa, Macrophominaphaseolina, Aspergil cantly compromise the structure and function of the enzyme. 10 luskawachii, Aspergillusaculeatus, Orpinomycesjoyonii and Such techniques include, but are not limited to, site-directed Acidothermuscellulolyticus. Table 3 provides the SEQ ID mutagenesis, cassette mutagenesis, random mutagenesis, NO: and database accession number for the amino acid including that conducted on isolated DNA or by exposing a sequences of wild-type, nativeFamily 5 cellulases from these microorganism to a mutagen, Such as UV light, synthetic organisms. oligonucleotide construction, cloning, Sub-cloning, amplifi 15 Modified Family 5 cellulases of the invention may have cation by PCR, in vitro synthesis and other genetic engineer conserved R130, H174, E217, E218, H288, Y290 and E329 ing techniques. It will be understood that the modified Family amino acids and exhibit greater than about 20% or greater 5 cellulase may be derived from any suitable Family 5 cellu than about 30% sequence identity with the TrCel5A catalytic lase. That is, it may be derived from a naturally-occurring or domain (amino acids 71-397 of SEQID NO:2). “wild-type Family 5 cellulase or from a Family 5 cellulase In another embodiment of the invention, the modified Fam that already contains otheramino acid Substitutions, deletions ily 5 cellulase has greater than about 30% sequence identity, or insertions. greater than about 40% sequence identity, greater than about By the term “wild-type Family 5 cellulase' it is meant a 50% sequence identity or greater than about 60% sequence Family 5 cellulase as found in nature that does not contain any identity with the TrCel5A catalytic domain (amino acids genetic alteration(s) introduced by molecular biology tech 25 71-397 of SEQ ID NO:2). niques. Family 6 Cellulase The position of the 363 mutation is determined by align Family 6 cellulases comprise two aspartic acid (D) amino ment with a wild-type Trichoderma reesei Family 5 enzyme, acids which may serve as catalytic amino acids. These aspar also referred to herein as “TrCel5A, the amino acid sequence tic acid amino acids are found at positions 175 and 221, as of which is provided in SEQID NO:2. By aligning the amino 30 determined by alignment with a wild-type Trichoderma acids to optimize the sequence similarity between the Family reesei enzyme. Most of the Family 6 cellulases identified thus 5 catalytic domains of cellulase enzymes, and by using the far are mesophilic; however, this family also includes ther amino acid numbering of TrCel5A as the basis for numbering mostable cellulases from Thermobifidafisca (TfGel6A and (also referred to herein as “TrCel5A numbering), the posi TfGel6B) and the alkalophilic cellulases from Humicolain tions of amino acids within other Family 5 cellulases can be 35 solens (HiCel6A and HiCel6B). Family 6 cellulases also determined relative to TrCel5A. The amino acid numbering is share a similar three dimensional structure: an alpha/beta based on the sequence of the mature, secreted protein, i.e., the barrel with a central beta-barrel containing seven parallel protein sequence after removal of the secretion signal peptide beta-strands connected by five alpha-helices. The three or leader peptide. dimensional structures of several Family 6 cellulases are Additional mutations may be introduced into the modified 40 known, such as TrCeléA, Thermobifidafisca endo-beta-1,4- Family 5 cellulase, provided that Such mutations do not sig glucanase CelóA, Humicolainsolens cellobiohydrolase nificantly compromise the structure and function of the CeléA, Humicolainsolens endo-beta-1,4-glucanase CeloB enzyme. As would be appreciated by those of ordinary skill in and Mycobacterium tuberculosis H37RV Cel6A. the art, but without being limiting in any manner, additional As used herein, the term “Family 6 cellulase' or “Celé” mutations may be introduced in regions of low sequence 45 encompasses a carbohydrate active cellulase enzyme that conservation among Family 5 cellulases. contains aglycohydrolase (GH) Family 6 catalytic domain The modified Family 5 cellulase of the invention may that is classified under EC 3.2.1.91 or EC 3.2.1.4. The term contain amino acid modifications “consisting essentially of encompasses any carbohydrate active enzyme that exhibits at the amino acid substitution at position 363. By this it is least hydrolysis of (1-4)-beta-D-glucosidic linkages in cellu meant that the modified Family 5 cellulase contains no more 50 lose linkages using an inverting mechanism, including than 20 other amino acid modifications in its sequence rela enzymes with conserved aspartic acid amino acids found at tive to a corresponding wild-type Family 5 cellulase. In positions 175 and 221 (based on alignment with Trichoderma another example of the invention, the modified Family 5 reesei CeléA amino acid numbering; SEQID NO:3). cellulase contains no more than 15 other amino acid modifi According to one embodiment, the Family 6 cellulase has cations, no more than 10 otheramino acid modifications or no 55 60% to 100% amino acid sequence identity to amino acids more than 5 other amino acid modifications in its sequence. 83-447 of SEQID NO:3 (Trichoderma reesei Family 6 cel As mentioned previously, such additional amino acid modi lulase), or, in a further embodiment, 65% to 100% amino acid fications may be introduced at non-conserved positions in the sequence identity to amino acids 83-447 of SEQ ID NO:3. amino acid sequence. Without being limiting in any manner, For example, the Family 6 cellulase may comprise an amino Such modifications are typically amino acid substitutions. 60 acid sequence that is 60, 65, 70, 75, 80, 85,90, 95 or 100% The additional amino acid modification(s), including, but identical to amino acids 83-447 of SEQID NO:3. not limited to amino acid substitutions, may be introduced by Table 4 below provides a representative list of known Fam standard molecular biology techniques such as random ily 6 cellulases and FIG. 9 shows an amino acid sequence mutagenesis, site-directed mutagenesis or directed evolution. alignment among the cellulases provided in the table. The Representative examples of Family 5 cellulases that do not 65 percent sequence identities of the Family 6 cellulases of Table contain an alanine at position 363 of the wild-type sequence 4 are determined using the procedure described previously for (TrCel5A numbering) and that can be modified in accordance the Family 45 cellulases. US 9.238,806 B2 15 16 TABLE 4 Family 6 cellulase family members and sequence identity with the TrCeléA catalytic domain (%). GenBank Percent identity with amino Accession SEQID acids 83-447 of TrCeléA Organism Number NO: (SEQID NO:3) Trichoderma reesei AAA34210. 3 1OO.O Hypocrea koningii AAKO1367. 79 98.9 Trichoderma viride CICC 13038 AAQ76094. 8O 98.9 Hypocrea koningii 3.2774 ABFS62O8. 81 98.1 Hypocrea koningii AS3.2774 ABG48766. 82 97.8 Trichoderma parceramosum AAUOS379.2 83 97.8 Aspergillus nidulans FGSC A4 ABF50873. 84 72.4 Aspergillus niger CBS 513.88 CAK41068. 85 72.4 Aspergillus oryzae RIB 40 BAE64227. 86 67.8 Aspergillus niger CBS 513.88 CAK39856. 87 67.7 Acremonium cellulolyticus Y-94 AAE50824. 88 67.3 Taiaromyces emersonii AAL781 65.2 89 66.8 Gibbereia zeae K59 AAQ72468. 90 66.1 Fusarium oxysportin AAA65585. 91 66.1 Neurospora crassa OR74A CAD70733. 92 65.9 Aspergillus nidulans FGSC A4 EAA65866. 93 65.5 Aspergilius tubingensis CAHOS675. 94 65.5 Magnaporthe grisea 70-15 XP 360146.1 95 65.4 Chaetomium thermophilum CAHOS669. 96 65.1 Chaetomium thermophilum CT2 AAW64927.1 97 6S.O Stilbeiia annulata CAHOS678. 98 64.9 Humicoia insolens BAB391,54. 99 63.7 Humicoia insolens 1BVW-8 1OO 63.1 Cochliobolus heterostrophus C4 AAM76664.1 101 59.6 Agaricus bisporus D649 AAASO607. 102 57.7 Polyporus arcularius 69B-8 BAF8O327. 103 57.1 Leniinitia edodes Stamets CS-2 AAK95.564. 104 56.3 Leniinitia edodes L54 AAK28357. 105 56.0 Mailbranchea cinnamonea CAHOS679. 106 S4.9 Phanerochaete chrysosporium AAB32942. 107 S4.9 Voivarielia voivacea AAT640O8.1 108 53.8 Chrysosporium lucknowense AAQ38151 109 49.5 Pleuroius Saior-Caiut AAL15037 110 47.2 Trametes versicolor AAF3S251. 111 47.0 Neurospora crassa OR74A XP 3233.15.1 112 46.8 Magnaporthe grisea 70-15 XP 362054.1 113 45.1 *Protein databank entry

The Family 6 cellulase may be a naturally-occurring or 40 Measurement of the Specific Depilling Activity of the wild-type Family 6 cellulase or a modified Family 6 cellulase. Enzyme Mixture By “modified Family 6 cellulase', it is meant a Family 6 The depilling composition of the present invention com cellulase that contains one or more genetic alteration, intro prises an enzyme mixture, which enzyme mixture comprises duced by molecular biology techniques, which do not signifi 45 (i) a Family 45 cellulase enzyme component; and (ii) at least cantly compromise the structure and function of the enzyme. one or more additional cellulase enzyme components that are Such techniques include, but are not limited to, site-directed selected from the group consisting of a Family 5 cellulase, a mutagenesis, cassette mutagenesis, random mutagenesis, Family 6 cellulase, and a combination thereof, wherein said including that conducted on isolated DNA or by exposing a enzyme mixture is enriched in the Family 45 cellulase and the microorganism to a mutagen, Such as UV light, synthetic 50 Family 5 and/or Family 6 cellulase components relative to a oligonucleotide construction, cloning, Sub-cloning, amplifi reference enzyme mixture. cation by PCR, in vitro synthesis and other genetic engineer By "enriched it is meant that the amount of the of the ing techniques. As would be appreciated by those of ordinary Family 45 and Family 5 and/or Family 6 cellulases, is skill in the art, but without being limiting in any manner, increased relative to all of the other cellulase components mutations introduced in regions of low sequence conserva 55 present in the enzyme mixture relative to those present in a tion among Family 6 cellulases are expected to have a lower reference or parental enzyme mixture. This may be achieved probability of reducing or altering the catalytic activity of the by increasing the amounts of the Family 45 and Family 5 enzyme. and/or Family 6 cellulases present in the enzyme mixture and/or decreasing the amounts of one or more other cellulase It will be understood that a modified Family 6 cellulase 60 components present in the reference enzyme mixture. may be derived from any suitable Family 6 cellulase. That is, By “reference enzyme mixture' it is meant an enzyme it may be derived from a naturally-occurring or “wild-type' mixture comprising the same Family 45 cellulase component Family 6 cellulase or from a Family 6 cellulase that already and the same Family 5 and/or Family 6 cellulase contains other amino acid Substitutions, deletions or inser component(s) as the enzyme mixture in the depilling compo tions. By the term “wild-type Family 46 cellulase' it is meant 65 sition but at naturally-occurring amounts. By “naturally a Family 6 cellulase that does not contain any genetic alter occurring amounts' it is meant the amount of the Family 45 ation(s) introduced by molecular biology techniques. and Family 5 and/or Family 6 cellulase components produced US 9.238,806 B2 17 18 by a parental microbe under essentially the same culturing some embodiments, the weight ratio of the Family 45 cellu conditions those used to produce the enzyme mixture of the lase component to the Family 6 cellulase component is depilling composition from a genetically modified microbe. between 5:1 and 1:1 (wt:wt) or any value therebetween. For For example, Such enzyme mixtures enriched in the Family example, the weight ratio of the Family 45 cellulase compo 45 cellulase and the Family 5 or Family 6 cellulase compo nent to the Family 6 cellulase component may be between 4:1 nents exhibit a specific depilling activity that is greater than and 1:1 or between 3:1 and 1:1 or between 2.7:1 and 1:1 or that exhibited by the corresponding reference enzyme mix between 2.7:1 and 1.25:1 (wt:wt) or any value therebetween. ture. Expression of the Cellulase Mixture in a Host Microbe In order to determine the specific depilling activity of the The present invention provides a genetically modified cellulase components in isolation and in combination, they 10 microbe expressing a Family 45 cellulase and one or more are typically purified using known techniques. Examples of additional cellulase enzyme component selected from a Fam purification techniques that can be utilized include affinity ily 5 cellulase, a Family 6 cellulase or a combination thereof. based purification technologies. Such technologies are well The host microbe may be any suitable yeast or a filamen known in the art and include any suitable method to selec tous fungus, such as those microbes that are members of the tively bind a component of a biological mixture to a solid 15 phylum Ascomycota. Genera of yeasts useful as host Support based on a highly specific biological interaction Such microbes for the expression of the enzyme mixture of the as that between antigen and antibody or enzyme and Sub present invention include Saccharomyces spp., Pichia spp. strate. Moreover, the purification can comprise fractionation Hansenula spp., Kluyveromyces spp., Yarrowia spp., and methods including selective precipitation Such as ammonium Arcula spp. Genera of fungi useful as microbes for the expres Sulfate precipitation, isoelectric precipitation, selective ther sion of cellulases of the present invention include Tricho mal denaturation or any other suitable method that selectively derma spp., Hypocrea spp., Aspergillus spp., Fusarium spp. precipitates the cellulase components. In another example, Humicola spp., Neurospora spp., Myceliopthora sp., Chrysos the purification methodology can comprise chromatographic porium spp., and Penicillium spp. In a most preferred embodi methods including gel filtration, size exclusion, anion ment, the host microbe is an industrial strain of Trichoderma exchange, cation exchange, gel electrophoresis, or other 25 reesei. chromatic separation methods known in the art for physically A parental host microbe is a microbe that is capable of the separating proteins. production and secretion of cellulase enzymes, but which The “specific depilling activity” as used herein, is deter exhibits wild-type or native copy number or expression of the mined as set forth in Example 1.6. The effectiveness of the identified genes that are increased in copy number or expres Family 45 cellulase in combination with the Family 5 cellu 30 sion in the genetically modified microbe. In the case of the lase or the Family 6 cellulase and the respective individual expression of a heterologous cellulase, the parental host cellulase components in removing small balls of fuzz, microbe is a strain that is capable of the production and referred to as pills, from fabric is measured by directly weigh secretion of cellulase enzymes, but which does not express, or ing released insoluble cellulose. The depilling is expressed as contain a copy of the gene encoding, the heterologous cellu the depilling activity per unit of protein (i.e., specific depill 35 lase. ing activity). Overexpression According to one embodiment of the invention, (i) the Overexpression of a cellulase enzyme refers to any state in Family 45 cellulase enzyme component and the Family 5 which that cellulase enzyme is caused to be expressed at an cellulase; or (ii) the Family 45 cellulase and the Family 6 elevated rate or level as compared to either (a) the endogenous cellulase enzyme component are present in the enzyme mix 40 expression rate or level of that same cellulase enzyme by the ture at a weight ratio that exhibits synergy in an assay that host microbe or (b) the expression rate or level of one or more measures specific depilling activity. other enzymes produced and secreted by the host microbe. As By “weight ratio that exhibits synergy', it is meant that the Such, overexpression of a cellulase enzyme may result from cellulase enzyme components are present in a weight ratio overexpression of a gene encoding the cellulase enzyme of that results in a specific depilling activity that is greater than 45 interest as well as a decrease in expression of one or more the sum of the specific depilling activity of each of said genes encoding one or more other enzymes produced and cellulase enzyme components assayed in isolation. The secreted by the host microbe. weight ratio is measured relative to the two cellulase enzyme Overexpression of a gene encoding a Family 45, Family 5 components in the enzyme mix. or Family 6 cellulase enzyme refers to any state in which such For example, in those embodiments in which the Family 45 50 gene is caused to be expressed at an elevated rate or level in and Family 5 cellulase components are included in the the host microbe as compared to the endogenous expression enzyme mixture at a weight ratio that differs from that present rate or level for that gene in the parental host microbe. In some in an enzyme mixture secreted by the parental strain, the examples, overexpression refers to an elevated transcription enzyme mixture will be characterized in that the Family 45 rate or level of an endogenous gene compared to the endog and Family 5 cellulase components, in combination, exhibit a 55 enous translation rate or level for that gene. In other examples, specific depilling activity that is greater than the sum of the overexpression refers to transcription of a gene encoding a Family 45 and Family 5 cellulase components assayed indi heterologous cellulase from another organism, which gene is vidually in isolation. introduced into a host cell. In some embodiments, the weight ratio of the Family 45 Overexpression of a gene encoding a Family 45, Family 5 cellulase component to the Family 5 cellulase enzyme com 60 or Family 6 cellulase enzyme may also refer to any state in ponent is between 0.20:1 and 9:1 (wt:wt) or any value ther which the rate or level of gene(s) encoding one or more other ebetween. For example, the weight ratio of the Family 45 (cellulase) enzymes produced and secreted by the host cellulase component to the Family 5 cellulase enzyme com microbe is reduced relative to the rate or level of the same ponent may be between 0.20:1 and 5:1 or between 0.25:1 and gene(s) in the parental host microbe. 4:1 or between 0.25:1 and 3:1 or between 0.25 and 2:1 or 65 A Family 45, Family 5 or Family 6 cellulase will be con between 0.25:1 and 1.5:1 or between 0.25 and 1.25:1 or sidered as being overexpressed by a modified microbe if the between 0.25 and 1.0:1 (wt:wt), or any value therebetween. In Family 45, Family 5 or Family 6 cellulase is produced at US 9.238,806 B2 19 20 higher levels by the modified microbe than by a parental host genome of the host microbe so that it integrates into the host microbe at essentially the same fermentation conditions. For genome. Alternatively, the genetic construct may not contain example, the Family 45, Family 5 or Family 6 cellulase may any polynucleotide sequences that direct sequence-specific be produced at an amount that is greater than about 1.1-fold to recombination into the host genome. In Such cases, the con about 50-fold, or any amount therebetween, than is produced struct may integrate by random insertion through non-ho by the parental host at essentially the same fermentation mologous end-joining and recombination. Alternatively, the conditions. For example, the Family 45 cellulase may be construct may remain in the host in non-integrated from, in produced at an amount that is greater than 1.1-, 1.5-, 2-, 5-, which case it replicates independently from the host 10- 20-, 30-, 40- or 50-fold higher, or an amount therebe microbe's genome. tween, than the amount produced by the parental host 10 The genetic construct(s) may further comprise a selectable microbe at essentially the same fermentation conditions. marker gene to enable isolation of a genetically modified Preferably, the Family 45 cellulase is produced at an amount microbe transformed with the construct as is commonly that is at least 1.1-fold more than the parental host at the same known to those of skill in the art. The selectable marker gene fermentation conditions. may conferresistance to an antibiotic or the ability to grow on In at least some embodiments of the present invention, the 15 medium lacking a specific nutrient to the host organism that increase or decrease in copy number or expression of a gene otherwise could not grow under these conditions. The present can be produced by any of various genetic engineering tech invention is not limited by the choice of selectable marker niques. As used herein, the term genetic engineering tech gene, and one of skill in the art may readily determine an nique refers to any of several well-known techniques for the appropriate gene. For example, the selectable marker gene direct manipulation of an organism's genes. For example, may confer resistance to hygromycin, phleomycin, kanamy gene knockout (insertion of an inoperative DNA sequence, cin, geneticin, or G418, or may complement a deficiency of often replacing the endogenous operative sequence, into an the host microbe in one of the trp, arg, leu, pyra, pyr, ura3. organisms chromosome), gene knock-in (insertion of a pro ura5, his, orade genes or may confer the ability to grow on tein-coding DNA sequence into an organism's chromosome), acetamide as a sole nitrogen source. and gene knockdown (insertion of DNA sequences that 25 The genetic construct may further comprise other nucleic encode antisense RNA or small interfering RNA, i.e., RNA acid sequences as is commonly known to those of skill in the interference (RNAi)) techniques are well known in the art. art, for example, transcriptional terminators, nucleic acid Methods for decreasing the expression of a gene also include sequences encoding peptide tags, synthetic sequences to link partial or complete deletion of the gene, and disruption or the various other nucleic acid sequences together, origins of replacement of the promoter of the gene Such that transcrip 30 replication, and the like. The practice of the present invention tion of the gene is greatly reduced or even inhibited. For is not limited by the presence of any one or more of these other example, the promoter of the gene can be replaced with a nucleic acid sequences. weak promoter, as exemplified by U.S. Pat. No. 6,933,133, The genetic construct may be introduced into the host which is incorporated by reference herein in its entirety. As microbe by any number of methods known by one skilled in used herein, a gene deletion ordeletion mutation is a mutation 35 the art, including but not limited to, treatment of cells with in which part of a sequence of the polynucleotide sequence CaCl2, electroporation, biolistic bombardment, PEG-medi making up the gene is missing. Thus, a deletion is a loss or ated fusion of protoplasts (e.g. White et al., WO 2005/ replacement of genetic material resulting in a complete or 093072, which is incorporated herein by reference). After partial disruption of the sequence of the DNA making up the selecting the recombinant strains, such strains may be cul gene. 40 tured in Submerged liquid fermentations under conditions According to one embodiment of the invention, overex that enable the expression of the Family 45, Family 5 and/or pression of a gene encoding the Family 45, Family 5 and/or Family 6 cellulase enzymes. Family 6 cellulase enzyme is achieved by introducing into a Depending on the host strain and the regulatory sequences host microbe one or more genetic construct(s) comprising a present in the genetic construct, expression levels can be polynucleotide sequence(s) encoding the cellulase enzyme(s) 45 modulated by adjusting one or more parameters of the fer that is to be overexpressed. The polynucleotide sequence(s) mentation process used to produce the cellulase enzymes encoding the cellulase enzyme(s) may be operably linked to from the host microbe including, but not limited to, the carbon regulatory sequences that direct the expression and secretion source, the temperature of the fermentation, or the pH of the of the cellulase enzyme(s), including: (i) a polynucleotide fermentation. Yet another means for adjusting expression lev sequence encoding a secretion signal peptide from a secreted 50 els of cellulase involves the modification of cellulase secre protein that may be endogenous or heterologous to the host tion pathways or modification of cellulase transcriptional cell; and (ii) a constitutive or regulated promoter derived from and/or translational regulation systems and/or post-transla a gene that is highly expressed in the host microbe under tional protein maturation machinery (e.g. transcription fac industrial fermentation conditions. In addition, a translational tors, protein chaperones). enhancer may be added to increase protein translation. These 55 Homologous and Heterologous Expression regulatory sequences may be derived from one or more genes, In some embodiments, the Family 45, Family 6 and/or including, but not limited to, the gene encoding the cellulase Family 5 cellulase component(s) in the enzyme mixture is enzyme to be expressed (provided that these regulatory endogenous to the host cell. In other embodiments, the Fam sequences are functional in the host cell). Moreover, multiple ily 45, Family 6 and/or Family 5 cellulase component is copies of the genetic construct(s) comprising a polynucle 60 heterologous or exogenous to the host cell. otide sequence(s) encoding the cellulase enzyme(s) may be For purposes herein, a heterologous or exogenous cellulase introduced into the microbe, thereby increasing expression enzyme refers to an enzyme that is encoded by a gene derived levels. Changes in expression can also be achieved by from a species that is distinct from the host microbe. An mutagenesis and selection of strains with desired expression endogenous or homologous cellulase enzyme refers to an levels. 65 enzyme that is encoded by a gene derived from the same The genetic construct may comprise other polynucleotide sequences as the host microbe. Thus, in some examples, a sequences that allow it to recombine with sequences in the homologous or heterologous cellulase enzyme is encoded by US 9.238,806 B2 21 22 a polynucleotide sequence that is derived from a species that molecular Solutes Such as unconsumed components of the is, respectively, the same as or different from the species of fermentation medium may be removed by ultra-filtration. The the host microbe expressing the cellulase enzyme, as well as enzyme mixture may be concentrated, for example, by evapo recognized anamorphs, teleomorphs or taxanomic equiva ration, precipitation, sedimentation or filtration. Chemicals lents of the host microbe. As is appreciated by one of skill in Such as glycerol. Sucrose, Sorbitol and the like may be added the art, the amino acid sequence of a homologous or heter to stabilize the cellulase enzyme. Other chemicals, such as ologous cellulase enzyme may be naturally-occurring (i.e., as Sodium benzoate or potassium Sorbate, may be added to the it is found in nature when produced by the Source organism) cellulase enzyme to prevent growth of microbial contamina or may contain one or more amino acid insertions, deletions tion. or Substitutions relative to the naturally-occurring amino acid 10 sequence as a result of genetic manipulation, adaptation or Treatment of Cellulose-Containing Goods. Using the Enzyme classical mutagenesis causing changes in the polynucleotide Mixture sequence encoding said heterologous cellulase enzyme. The enzyme mixture of the present invention is used in the Although expression from a single host microbe has been depilling of “cellulose-containing goods’. described, the Family 45, Family 6 and/or Family 5 cellulases 15 The term "cellulose-containing goods’ refers to fabrics, may be expressed individually or in sub-groups from differ either as piece goods or goods sewn into garments or yarn, ent strains of one or more host microbes. For example, it is comprising cotton or non-cotton containing fibers. The cel contemplated that the Family 45, Family 6 and/or Family 5 lulose-containing goods may be treated with the enzyme mix cellulases may be expressed individually or in Sub-groups ture of the invention either before or after dyeing and with or from different strains of a single host microbe. without a resinous finish. The term encompasses natural cel After selecting the recombinant host strains overexpress lulosics and manmade cellulosics. Manmade cellulose con ing the Family 45, Family 6 and/or Family 5 genes, they may taining fabrics include regenerated fabrics, such as rayon, that be cultured in Submerged liquid fermentations under condi are well known in the art. The term excludes fabrics that are tions that induce the expression of the cellulases, as discussed not prone to pilling, Such as denim fabrics. below. 25 As used herein, the term “depilling” refers to the use of the Production of the Cellulase Enzyme Mixture from the Host enzyme mixture of the present invention in a controlled Microbe hydrolysis of cellulosic fibres in order to modify the surface Submerged liquid fermentations of microorganisms are of the cotton goods in a manner that clears the Surface struc typically conducted as a batch, fed-batch or continuous pro ture by reducing fuZZing. Such treatment can prevent pilling, cess. In a batch process, all the necessary materials, with the 30 exception of oxygen for aerobic processes, are placed in a or improve fabric handle like softness and smoothness, which reactor at the start of the operation and the fermentation is can result in clarification of colour and/or improve moisture allowed to proceed until completion, at which point the prod adsorbability and dyeability. uct is harvested. A batch process for producing the enzyme Depilling treatment may be carried out during the manu mixture of the present invention may be carried out in a 35 facturing process or in Subsequent laundering. In either case, shake-flask or a bioreactor. treatment is typically carried out by adding cotton goods to a In a fed-batch process, the culture is fed continuously or rotating horizontal or vertical drum jet dryer, washing sequentially with one or more media components without the machine, or other device that contains the fabric, water, buffer removal of the culture fluid. In a continuous process, fresh and cellulase enzyme, while providing agitation and shear to medium is Supplied and culture fluid is removed continuously 40 the fabric, including loose fibrils. Detergents and Surfactants at Volumetrically equal rates to maintain the culture at a may also be added during the depilling treatment. After treat steady growth rate. ment, the fabric is removed from the machine and dried. Fermentation medium typically comprises a carbon When depilling takes place in a typical manufacturing Source, a nitrogen source and other nutrients, vitamins and process, the treatment time may be between about 15 to about minerals which can be added to the fermentation media to 45 120 minutes; treatment temperature may be about 35°C. to improve growth and enzyme production of the host cell. The about 60° C.; the weight ratio of liquor to fabric may be various media components may be added prior to, simulta between about 2.5:1 and about 10:1; and the pH may be about neously with or after inoculation of the culture with the host 4.0 to about 6.0. When depilling takes place in a typical cell. laundering, the treatment time is about 10 to about 60 min When producing the cellulase enzyme mixture of the 50 utes, the treatment temperature is about 20° C. to about 70° present invention, the carbon Source may comprise a carbo C., the weight ratio of liquor to fabric is between about 2.5:1 hydrate that will induce the expression of the cellulase and about 10:1, and the pH is about 4.0 to about 9.5 or about enzyme mixture from the genetically modified microbe. For 4.0 to about 6.0. example, the carbon source may comprise one or more of The amount of cellulase mixture used to depill depends on cellulose, cellobiose, Sophorose, and related oligo- or poly 55 the concentration of active protein in the cellulase mixture, saccharides known to induce expression of cellulases. the amount of cotton goods being treated, the desired degree In the case of batch fermentation, the carbon source may be of depilling, the desired time of treatment, and other param added to the fermentation medium prior to or simultaneously eters well-known to those of ordinary skill in the art. When with inoculation. In the cases of fed-batch or continuous used for depilling in a typical manufacturing process, an operations, the carbon source may be supplied continuously 60 example of a dose of cellulase is between about 0.1 and about or intermittently during the fermentation process. For 7 g of enzyme protein per kilogram of fabric and more pref example, the carbon feed rate may be between 0.2 and 4.0 g erably between about 0.5 g and about 4 g of enzyme protein carbon/L of culture/h, or any amount therebetween. per kilogram of fabric. When used for depilling in a typical Following fermentation, the fermentation broth containing laundering, the preferred amount of cellulase is generally the cellulase enzyme mixture may be used directly, or the 65 between about 0.01 g and about 3 g of enzyme protein per cellulase enzyme mixture may be separated from the host kilogram of fabric and more preferably between about 0.05g cells, for example by filtration or centrifugation. Low and about 2.5g of enzyme protein per kilogram of fabric. US 9.238,806 B2 23 24 One option for controlling the action of the enzyme is to and terminated by the terminator sequence of an N. crassa destroy the enzyme after treatment by heating the solution, orotidine-5'-monophosphate decarboxylase (pyra) gene adding chemicals to destroy enzyme activity or by drying the (Tpyr4). The expression cassette contains a T. reesei cel7a cotton goods. promoter (Pcel7a) as a part of the 5' flank of the Trcel7a Detergent compositions of the present invention may be in targeting fragment, a T. reesei pre-mature Cel45A coding any form known in the art. This includes as a liquid diluent, in sequence (including the TrCel45A secretion signal) and a T. granules, in emulsions, in gels, or in pastes. When a Solid reesei celóa terminator (TCel6a) as shown in FIG. 4. The detergent composition is employed, the enzyme mixture is vector pP7a-TrCel45A-hph-TV was linearized with Psi I typically formulated as granules. before transformation. 10 The transformation vector, pP7a-xyn2ss-Cel45A-hph-TV EXAMPLES (FIG. 5), also contains the E. coli hygromycinphosphotrans ferase gene (hph) as a selectable marker. The transcription of Example 1 the hph gene is driven by the phophoglycerate kinase pro Generation and Characterization of Cel45A 15 moter and terminated by a pyra(N. crassa orotidine-5'-mono Overexpressing Strains phosphate decarboxylase) terminator. The expression cas sette contains a T. reesei cel7a promoter (Pcel7a) as a part of Example 1.1 the 5' flank of the cel7a targeting fragment, a T. reesei Xyla nase 2 secretion signal coding sequence (Xyn2SS), a T. reesei Host Trichoderma reesei Strains for the mature Cel45A coding sequence and a T. reesei celóa termi Overexpression of Cel45A Cellulase nator (TCel6a) as shown in FIG. 5. The vector, pP7a-xyn2ss Cel45A-hph-TV, was linearized with Psil before transforma Theparental Trichoderma reesei strain used for the over tion. expression of TrCel45Acellulase is P998A (described in co The transformation vector, pP6a-6Ass-Cel45A-ble-TV pending and co-owned U.S. patent application No. 61/312, 25 (FIG. 6), contains an Shble bleomycin resistance gene as a 864). The strain P998A was generated by the transformation selectable marker. The Shble gene encodes the Streptoallotei of a BTR213 uridine auxotroph strain with a transformation chushindustanus bleomycin resistance protein, ShBle, which vector (Pc/x-cel5A-G363 A-pyra-TV) designed to express confers resistance to bleomycin, Zeocin and phloemycin. The high levels of Cel5A-G363A (SEQ ID NO: 1) enzyme and transcription of the ShBle gene from the selection cassette is restore prototrophic growth in the absence of uridine (see 30 driven by a tefl (translation elongation factor 1) promoter U.S. patent application No. 61/312.864, supra). The strain (Ptef1) and terminated by a cel7a terminator (Tcel7a). The BTR213 is a derivative of strain RutC30 (ATCC #56765) that expression cassette contains a T. reesei celóa promoter was isolated as a high cellulase producing derivative of the progenitor strain QM6A (Montencourt et al., Proc. 3" (Pcel6a), a T. reesei Cel6A secretion signal coding sequence Annual Symp. On Fuels from Biomass, Golden Colorado, 35 (CeléASS), a Treesei mature Cel45A coding sequence and a 1979, pp. 85-89). Further cellulase hyper-producing strains Treesei cel45a (Tcel45a) terminator as shown in FIG. 6. The were generated from RutC30 by random mutation and/or vector, pP6a-6Ass-Cel45A-ble-TV, was linearized with PacI selection. Strain M2C38(ATCC 74252) was isolated based on before transformation. its ability to produce larger clearing Zones than RutC30 on minimal media agar containing 1% acid Swollen cellulose 40 Example 1.3 and 4 g L' 2-deoxyglucose. Subsequently, M2C38 was sub jected to further random mutagenesis and strain BTR213 was Generation of TrCel45A Overexpressing T. reesei isolated by selection on lactose media containing 0.2 g/mL Strains carbendazim. A uridine auxotroph of BTR213, BTR213aux, was obtained through selection of mutants spontaneously 45 The strains overexpressing TrCel45A from a single inte resistant to 0.15% w/v 5-fluoroorotic-acid (FOA). The pro grated transgene copy were generated by transformation of totrophy was restored by transformation with a heterologous the parental P998A strain with transformation vectors, pP7a Neurosporacrassa gene pyra encoding orotidine-5'-mono xyn2ss-Cel45A-hph-TV or pP7a-TrCel45A-hph-TV. phosphate decarboxylase used as a selection marker to gen designed to express high levels of native Treesei Cel45A and erate the Strain P998A. 50 target the native Trcel7a locus. The transformation vector was introduced by poly-ethylene glycol (PEG) mediated transfor Example 1.2 mation. The P998A strain (5x10 spores) was plated onto sterile cellophane overlaid on Potato Dextrose agar (PDA) T. reesei Transformation Vectors for Overexpression and the plates were incubated for 20 hours at 30°C. to facili of TrCel45A 55 tate spore germination and mycelial growth. Cellophane discs with mycelia were transferred to 10 mL of a protoplasting Three TrCel45A expression vectors were used for Treesei Solution containing 7.5 g/LDriselase and 4 g/L beta-gluca transformations. All vectors were designed to target the native nase (InterSpex Products Inc., Cat. Nos. 0465-1 and 0439-2, Trcel7a locus. To facilitate targeting, sequences adjacent to respectively) in 50 mM potassium phosphate buffer, pH 6.5 the 5' and 3' ends of the native Trcel7a gene amplified from 60 containing 0.6 Mammonium sulfate (Buffer P). The mycelial BTR213 genomic DNA were inserted into the transformation mat was digested for 5 hours by gentle agitation at 60 rpm. vectors so as to flank the expression and selection cassettes Protoplasts were separated from the undigested mycelia by (FIGS. 4-6). filtration through sterile No. 30 MIRACLOTHTM and col The transformation vector, pP7a-TrCel45A-hph-TV (FIG. lected into a sterile 50 mL round-bottom centrifuge tube and 4), contains the E. coli hygromycinphosphotransferase gene 65 recovered by centrifugation at 1000-1500x g for 10 min at (hph) as a selectable marker. The transcription the hph geneis room temperature. Protoplasts were washed with 5 mL of driven by a Treeseiphophoglycerate kinase promoter (Ppgk) Buffer P and centrifuged again at 1000-1500x g for 10 minat US 9.238,806 B2 25 26 room temperature. Protoplasts were resuspended in 1 mL of TABLE 6 STC buffer (1.2M sorbitol, 10 mM CaCl, 10 mMTris-HCl, pH 7.5). For transformation, 0.1 mL of resuspended protoplasts was The composition of micro-culture media combined with 0.01 mg of transformation vector linearized with Psil restriction enzyme and 0.025 mL of PEG solution Component g/L (25% PEG 3350, 50 mM CaCl, 10 mMTris-HCl, pH 7.5). After incubation in an ice water bath for 30 min, 1 mL of the KH2PO 8 PEG solution was added and the mixture incubated for 5 min (NH4)2SO 12.7 MgSO4·7H2O 4 at room temperature. The resultant transformation mix was 10 diluted with 2 mL of STC buffer and the entire mix was added CaCl2.H2O 1.02 to 50 mL of molten MMSSagar media (Table 5 below) cooled Corn steep liquor 5 to about 47°C. and poured into large plates. The plates were incubated at 30°C. overnight and an overlay of MMSS media CaCO 2O supplemented with 60 U/mL of hygromycin was added for Carbon Source** 35 the selection of transformed cells. The plates were further 15 Trace elements 2 mLL incubated at 30° C. until colony growth was visible. Trans *Trace elements solution contains 5 g/L FeSO4·7H2O; 1.6 g/L MnSO4·H2O; and 1.4 g/L formants were transferred to individual plates containing ZnSO4·7H2O. PDA agar Supplemented with hygromycin and allowed to Glucose, Solkafloc, lactose, cellobiose, sophorose, corn syrup, or Avicel. The carbon sporulate. Spores were collected and plated at high dilution source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the on PDA-hygromycin media to isolate homokaryon transfor remaining media initially or through the course of the fermentation, mants, which were then plated onto PDA to allow for growth Cultures were grown for 6 days at 30°C. with shaking at and Sufficient sporulation to inoculate the screening cultures 250 rpm. The biomass was separated from the growth media described below. One transformant containing a single of the containing the secreted protein by centrifugation at 12,000 pP7a-xyn2ss-Cel45A-hph-TV vector integrated ectopically rpm. The protein concentration was determined using the into the P998A genome was selected for further transforma 25 tion. Bio-Rad Protein Assay (Cat. No. 500-0001). The relative The strains overexpressing TrCel45A from two integrated abundance (in weight % of total secreted protein) of transgene copies were generated using P1498Seas a parental TrCel45A, TrCel5A and TrCel7A was determined by ELISA. strain. This strain was transformed with the transformation For ELISA measurements the enzymes were first desalted vector, pP6a-6Ass-Cel45A-ble-TV, designed to express high 30 using BioSpin columns (Biorad) following manufacturer levels of native T. reesei Cel45A and target the cel7a locus. instructions and total protein concentration was determined This vector was also introduced by poly-ethylene glycol using a BCA kit (Sigma) with a bovine serum albumin (PEG) mediated transformation as described above. The (Sigma) control. The same assay was used to determine the transformants were selected on PDA media containing 100 concentration of purified protein standards. Culture Superna ug/mL of phleomycin. 35 tants and purified component standards were diluted to 0.01 10 ug/mL in phosphate-buffered saline, pH 7.2 (PBS) and TABLE 5 incubated overnight at 4°C. in microtitre plates (Costar EIA #9018). These plates were washed with PBS containing 0.1% Minimal medium (MM) agar 40 Tween-20 (PBS/Tween) and then incubated in PBS contain Amount ing 1% bovine serum albumin (PBS/BSA) for 1 hour at room Component per L. temperature. Blocked microtitre wells were washed with KH2PO 10 g PBS/Tween. Rabbit polyclonal antisera specific for (NH4)2SO 6g TrCel45A, TrCel5A or TrCel7A were diluted in PBS/BSA, NaCitrate2HO 3g added to separate microtitre plates and incubated for 2 hours FeSO4·7H2O 5 mg 45 MnSOHO 1.6 mg at room temperature. Plates were washed and incubated with ZnSO7H2O 1.4 mg a goatanti-rabbit antibody coupled to horseradish peroxidase CaCl2HO 2 mg (Sigma #A6154), diluted 1/2000 in PBS/BSA, for 1 hour at Agar 20 g 20% Glucosefs. 50 mL. room temperature. After washing, tetramethylbenzidine was 1M MgSO7H2O 4 mL 50 added to each plate and incubated for 30 minutes at room pH to 5.5 temperature. The absorbance at 360 nm was measured in each MMSS agar contains the same components as MMagar plus 1.2M sorbitol, 6.6 g/LYNB well and converted into a protein concentration using stan (Yeast Nitrogen Base without Amino Acids from DIFCO Cat, No. 291940) and 1.92 g/L dard curves for each component. The abundance of amino acids (-Ura DO Supplement from Sigma Cat. No. Y1501-20G). TrCel45A, TrCel5A and TrCel7A was expressed as the mass 55 percent of each component as a fraction of total Secreted Example 1.4 protein (data not shown). Characterization of TrCel45A Overexpressing T. The strains P1467Ve, P1498Se., P1489Ct, P1489Et, reesei Strains P1554ABt, P1554Te and P1554Le were selected for further 60 analysis in 14 L pilot fermentation as they produced higher To test the production of the TrCel45A, TrCel5A and levels of TrCel45A enzymethan the parental strains BTR213 TrCel7A proteins, spores of Trichoderma transformants and and/or P998A and maintained high levels of TrCel5A-G363A the parental strain grown on PDA plates were suspended in enzyme. In addition to the increased expression of TrCel45A sterile water and about 10-10° spores per mL were used to and TrCel5A-G363 A, strains P1489Ct, P1489Et and inoculate each micro-culture in 24-deepwell plates. The com 65 P1554ABt also are deficient in the production of native ponents present in the micro-culture media are provided in TrCel7A cellulase. The modifications introduced into Table 6. selected strains are shown in Table 7. US 9.238,806 B2

TABLE 7 Modifications introduced into Selected Cel45A overexpressing strains. TrCel45A Parental TrCel5A- Secretion signal Selection marker Strain strain G363A TrCel7A 1 copy 2 copy 1 copy 2 copy P1467We P998A OE -- Cel45A NA hph NA P1498Se. P998A OE -- Xyn2 NA hph NA P1489Ct. P998A OE A Xyn2 NA hph NA P1489Et P998A OE A Xyn2 NA hph NA P1554ABt P1498Se. OE A Xyn2 Cel6A hph Shble P1554Te P1498Se. OE -- Xyn2 Cel6A hph Shble P1554Le P1498Se. OE -- Xyn2 Cel6A hph Shble OE-overexpressed, A - deletion, NA—not applicable

Example 1.5 TABLE 9 2O Initial Media for Fed-Batch Fermentations Enzyme Production in 14 L. Pilot Fermentation Component g/L Trichoderma spores of strains BTR213, P998A, P1467Ve, (NH4)2SO 2.2O KH2PO 1.39 P1498Se. P1489Ct, P1489Et, P1554ABt, P1554Te and 25 MgSO4·7H2O O.70 P1554Le grown on PDA media were suspended in sterile CaCl2HO O.185 Dry Corn Steep 6.OO water and transferred to 2 L, baffled Erlenmeyer flasks con Liquor taining 750 mL of liquid Berkley media (pH 5.5) supple Glucose 13.00 mented with 5.1 g/L of corn steep liquor powder and 10 g/L Trace elements 0.38 mLL glucose (Table 8). Flasks were incubated at 28°C. for 3 days 30 *Trace elements solution contains 5 g/L FeSO4·7H2O, 1.6g/L MnSOHO and 1.4 g/L using an orbital agitator (Model G-52 New Brunswick Sci ZnSO4·7H2O. entific Co.) running at 100 rpm. The biomass content of the culture broth was determined TABLE 8 using aliquots of 5-10 mL of broth that had been weighed, 35 vacuum filtered through glass microfiber filters, and oven Berkley Media for Flasks dried at 100° C. for 4 to 24 hours. The concentration of biomass was determined according to the equation below. Component g/L (NH4)2SO 10.4 dry filter paper and cake (g) - filter mass (g) KH2PO 2.0 40 Biomass (g/L) = X MgSO4·7HO O.31 wet sample-mass (g) CaCl2.H2O O.S3 Dry Corn Steep Liquor S.1 broth density (g/mL) x 1000 mL/L Glucose 10 Trace elements 1 mLL 45 The protein concentration of the culture filtrate was deter *Trace elements solution contains 5 g/L FeSO4·7H2O, 1.6 g/L MnSOHO and 1.4 g/L ZnSO4·7H2O. mined using the Bradford assay. Colour intensity changes in the Coomassie Brilliant Blue G-250 dye, that forms the basis The content of eachinoculum flask was transferred to a 14 of this assay, were quantified spectrophotometrically using L. pilot scale fermentation vessel (Model MF1 14 New Brun absorbance measurements at 595 nm. The standard assay swick Scientific Co.) containing 10 L of Initial Pilot Media 50 control used was a cellulase mixture of known composition having a pH of 5.5 (Table 9). The vessel was run in batch and concentration. The final filtrates for enzyme analysis mode until glucose in the media was depleted. At this point, were collected after 162-170 hours. the carbon source containing cellulase inducing carbohy The relative concentrations (in weight percent of total drates was added on a continuous basis from a stock that was secreted protein) of four cellulase components (TrCel7A, 55 TrCel6A, TrCel45A, TrCel5A) were determined by ELISA 35.5% w/v of solids dissolved in water. Peristaltic pumps using a component specific antibody as described above (EX were used to deliver the carbon source at a feed rate of 0.4 ample 1.4). The abundance of TrCeléA cellulase is similar in grams of carbon per liter culture per hour. Operational param all transformants and parental strains. The TrCel7A cellulase eters during both the batch and fed-batch portions of the run was not detected in transformants deleted in Trcel7a. Trans were: mixing by impeller agitation at 500 rpm, air sparging at 60 formants with ectopically integrated Trcel45a genetic con 8 standard liters per minute, and a temperature of 28°C. structs have similar TrCel7A levels as their parental strains. Culture pH was maintained at 4.0-4.5 during batch growth The relative abundance of TrCel5A in Trcel7a deleted trans and pH 4.0 during cellulase production using an automated formants increased compared to that of the parental strains or controller connected to an online pH probe and a pump transformants that still produce the native TrCel7A enzyme. enabling the addition of a 10% ammonium hydroxide solu 65 The abundance of TrCel45A increased in all transformants tion. Periodically, 100 mL samples of broth were drawn for possessing either the single or double integrated Trcel45a biomass and protein analysis. transgene copy (FIG. 7). US 9.238,806 B2 29 30 Example 1.6 pyra-TV, was linearized with EcoRI before transformation and was introduced by poly-ethylene glycol (PEG) mediated Depilling Activity of Enzyme Produced by T. transformation as described in Example 1.3. Transformants reesei Transformants containing at least one copy of integrated vector pChhiCel5A-pyra-TV were identified by the amplification For testing depilling activity, circles of 100% cotton flan of a 1.7 kb product from genomic DNA using primers specific nelette fabric with an approximate 4.5" diameter and weigh for the Treesei cel7a promoter and the Treesei celóa termi ing ~1.0-2.0 g were cut. The initial weight of each piece of nator and Subsequently confirmed by Southern blot analysis fabric was measured and recorded. Fabric circles were placed of genomic DNA using a probe specific for the Hicel5a cod in the jars fuzzy side up and 90 g of steel ball bearings and 50 10 ing region (data not shown). Integration of the vector into the mM citrate buffer, pH 5.0 were added into each jar. The Trcel7a locus was confirmed by Western blot analysis of enzymes were diluted to obtain 1-10 mg of the total protein filtrates from microcultures of transformants grown in cellu per jar. The diluted enzyme was added to the jars so that the lase-inducing medium (data not shown). Strain P579A was total liquid in each jar equaled about 15 g. The jars were identified as having one copy of the pChHiCel5A-pyra-TV incubated at 50° C. for 2 hours with shaking at ~180 rpm. 15 integrated into the Trcel7a locus. After incubation, 3 drops of 10 N. NaOH were added to the jars to stop the depilling reaction. Subsequently, the contents Example 2.2 of the jars were filtered and the fabric was returned to the jar. After addition of about 200 mL of deionized water, the jars T. reesei Transformation Vectors for Overexpression were shaken vigorously for about 15 seconds to release the of HiCel45A “fines' from the fabric and the fabric was discarded. The liquid was filtered, dried in a 100° C. oven for at least two The transformation vector, Pc/x-TrHicel45a-ble-TV (FIG. hours and the total weight of filter paper and released fibers 10) was used for the expression of the H. insolens Cel45A was recorded. The loss of fabric weight was calculated using gene (Hicel45A: SEQ ID NO:115). It contains the Shble equation below: 25 bleomycin resistance gene as a selectable marker. The Shble gene encodes the Streptoalloteichushindustanus bleomycin resistance protein, ShBle, which confers resistance to bleo (weight of the filter paper + collected fines) - mycin, Zeocin and phleomycin. The transcription of the S. weight of the filter paper hindustanus ShBle protein from the selection cassette is % weight loss = x 100% 30 initial weight of the fabric driven by a tefl (translation elongation factor 1) promoter (Ptef1) and terminated by a cel7a terminator (Tcel7a). The expression cassette contains a hybrid T. reesei cel7afxyn2 The depilling activity was compared to that of an enzyme promoter (Pc/x: cel7a promoter 5448-6643 nucleotides and mixture produced from strain BTR213 and assayed at pH 5.0 Xyn2 promoter 664-6817 nucleotides), a xylanase 2 secretion (set to a depilling activity of 1.0). 35 signal (X2ss), a T. reesei sequence optimized H. insolens As shown in FIG. 8, enzyme mixtures produced by over Cel45A coding sequence (Hicel45a; (SEQID NO:115) and a expression of TrCel45A and overexpression of the modified T. reesei cel7b terminator (Tcel7b) as shown in FIG. 10. TrCel5A-G363A protein resulted in an increased depilling activity relative to the enzymes produced by the P998A Example 2.3 parental strain. 40 Generation of T. reesei Strains Overexpressing Example 2 HiCel45A and HiCel5A Generation and Characterization of HiCMC3 and The strains overexpressing HiCel45A from a single inte HiCel45A Overexpressing Strains 45 grated transgene copy were generated by transformation of the HiCel5A overexpression strain P579A with the transfor Example 2.1 mation vector, Pcfx-Tricel45a-ble-TV. The transformation vector was introduced by poly-ethylene glycol (PEG) medi Expression of HiCel5A Cellulase in Treesei ated transformation as described in Example 1.3. 50 The HiCel5 overexpression strain P579A was generated by Example 2.4 the transformation of a M2C38uridine auxotroph strain with the pChHiCel5A-pyra-TV transformation vector (FIG. 9) Characterization of HiCel45A+HiCel5A designed to express the Hicel5a gene (SEQ ID NO:114), Overexpressing T. reesei Strains target the Trcel7a locus and restore prototrophic growth in the 55 absence of uridine. The Hicel5a gene (SEQ ID NO:114) To test the production of the HiCel45A, spores of Tricho encodes a Family 5 cellulase having the amino acid sequence derma transformants and the parental strain were grown in of SEQ ID NO: 74 (HiCMC3). A uridine auxotroph of microcultures as described in Example 1.4. Cultures were M2C38, M2C38aux, was obtained through selection of grown for 6 days at 30° C. with shaking at 250 rpm. The mutants spontaneously resistant to 0.15% w/v 5-fluoroorotic 60 biomass was separated from the growth media containing the acid (FOA). The transcription of the N. crassa gene pyra from secreted protein by centrifugation at 12,000 rpm. The protein the selection cassette is driven by its native promoter (Ppyr4) concentration was determined using the Bio-Rad Protein and terminator (Tpyra). The expression cassette contains a T. Assay (Cat. No. 500-0001). reesei cel7a promoter (Pcel7A) and secretion signal (as a part The presence of HiCel45A was determined by immunob of the 5' flank of the cel7a targeting fragment), a H. insolens 65 lotting. Approximately 2 ug of total protein from microcul mature Cel5A coding sequence (Hicel5a; SEQ ID NO:114) ture filtrates was fractionated on a 10% SDS-PAGE precast and a T. reesei celóa terminator. The vector, pChFIICel5A gel (BioRad) prior to Western hybridization with Cel45A US 9.238,806 B2 31 32 antibody (data not shown). Samples were mixed with sample Example 1.5. The expression of HiCel45A was determined buffer for a final concentration of 2% w/v SDS, 10% w/v. by immunoblotting using a Cel45A Subfamily A specific glycerol, 50 mMTris-HCl pH 6.8, 100 mM DTT, 0.05% bro antibody as described above (Example 2.4). A ~48 kDa band mophenol blue and 1.25% V/v B-mercaptoethanol. Samples was detected in both transformants but is absent in the parent comprised of 2 ug protein from microculturesbroth or 400ng Strain P579A. The abundance of HiCel45A is similar in both of PpHiCel45A purified from a 14L fermentation of a Pichia transformants (FIG. 11). pastoris strain expressing HiCel45A. Samples were boiled for 5 min prior to be loaded on the 10% precast Tris-HC1 gel Example 2.6 (Biorad). Following electrophoresis at 100 V for 10 min and 1 hr at 120V, gels were transferred to a PVDF membrane for 10 Depilling Activity of Cellulase Mixtures Enriched in Western hybridization. Transfer was done at 100V for 1.5 HiCel5A and HiCel45A+HiCel5A hours at room temperature with an ice pack. The membrane was then rinsed at room temperature as follows: 5 min The depilling activity of enzyme mixtures produced in 14 Milli-Q H.O. 20 min TTBS. The membrane was then incu L. fermentation from strains BTR213 P579A and P1622A, bated overnight at 4°C. in 20 ml of a 1:5 000 dilution of a 15 was determined as in Example 1.6, except that the depilling HiCel45A probing rabbit raised polyclonal antisera in HST reactions were carried out in 50 mM phosphate, 50 mM solution. Following the overnight incubation with the pri citrate buffer, pH 6.5 and the jars were shaken vertically for mary antibody, the membrane was rinsed at room temperature about 60 seconds at 750 rpm on a 2.54 cm span shaking table as follows: 2x5 min in TTBS, 1x5 min in HST, 2x5 min in to release the “fines' from the fabric. The depilling activity TTBS. The secondary antibody (Goat raised anti-Rabbit HRP was compared to that of an enzyme mixture produced from conjugated) was diluted 1:2000 in HST. 20 ml of the diluted strain BTR213 and assayed at pH 5.0 (set to a depilling solution was added to the membrane and incubated for 1 hour activity of 1.0). at room temperature. Following the incubation with the sec ondary antibody, the membrane was rinsed at room tempera As shown in FIG. 12, enzyme mixtures enriched in ture as follows: 3 times 5 mininTTBS, 10 minin HST, 3 times 25 HiCel45A and HiCel5A (from strain P1622A) show 5 min in TTBS, 5 min in TBS. The Western blot was devel increased depilling activity relative to an enzymes mixture oped using the SuperSignal West Dura extended duration enriched only in HiCel5A (the cellulase mixtures from paren substrate kit (Thermo Scientific) as per manufacturer's rec tal strain P579A) or to an enzyme mixture that is not enriched ommendation. The images were captured with animager with in either component (i.e., the cellulase mixtures from Strain no light, no filter for the chemiluminescence signal and white 30 BTR213). light, EtBr/UV filter for the molecular weight marker. A ~48 kDa band was detected in all transformants but not in the Example 3 parent strain P579A. Two strains, P1622A and P1622F, were chosen for fermentations in 14 L vessels. Purification of TrCel45A, TrCel5A-G363A. TrCel5A 35 and TrCel6A

Immunoblotting Solution Component g/L Example 3.1 Purification of Trichoderma reesei TrCel5A and TrCel6A TTBS Tris-base 2.42 (Tris buffered saline with NaCl 29.22 Tween-20). pH7.5 Tween-20 1 ml TrCel5A and TrCel6A were purified from a complete cel HST Tris-base 2.42 lulase mixture produced by T. reesei strain BTR213 as pH7.5 NaCl 58.44 described in U.S. Pat. No. 88,071,373. Tween-20 5 ml TTBS Tris-base 2.42 (Tris buffered saline) pH7.5 NaCl 29.22 Example 3.2 Purification of Trichoderma reesei 45 Cel45A A strain of Trichoderma reesei deleted in the genes encod TABLE 10 ing TrCel7A, TrCel6A and TrCel5A (strain P297J, described in WO2010/096931A1), was grown in submerged liquid fer Modifications introduced into selected HiCel45A overexpressing strains. 50 mentation under similar as those described in Example 1.5. HiCel45A The fungal cells were removed from the fermentation broth Parental secretion Selection by filtration across a glass microfiber filter containing a Har Strain strain HiCel5A TrCel7A signal marker borlite filter bed. TrCel45A was separated from other secreted P1622A P579A OE A Xyn2 ble proteins using anion exchange and size exclusion chromatog P1622F P579A OE A Xyn2 ble 55 raphy as described below. A 60 mL column of DEAE-Sepharose was equilibrated in OE-overexpressed, 10 mMTris/10 mMBis-Tris, pH 7.5 (Buffer 1). The cell-free A—deletion fermentation broth, equivalent to about 260 mg of total pro tein, was adjusted to Buffer 1 conditions and applied to the Example 2.5 60 column at 5 mL/min. The column was washed with 600 mL of Buffer 1, followed by 300 mL of 10 mMTris/10 mMBis-Tris, Production of Cellulase Mixtures Enriched in pH 6.5 (Buffer 2). These two steps elute a significant propor HiCel5A and HiCel45A+HiCel5A in 14 L Pilot tion of the non-TrCel45A protein from the column. A 900 mL Fermentation linear gradient of 0-150 mMNaCl was applied to the column 65 in a Buffer 2 background. Two major peaks elute during this Cellulase mixtures were produced from strains P579A, gradient. Peak 2 contains TrCel45A and some other proteins. P1622A and P1622F in 14 L fermentations as described in Fractions corresponding to Peak 2 were pooled and concen US 9.238,806 B2 33 34 trated using a stirred ultrafiltration cell (Amicon) and a 10 The supernatant containing TrCel5A-G363A was then kDa NMWL polyethersulfone membrane. separated by anion exchange chromatography using the same A concentrated preparation of Peak 2 was applied to a 170 procedure described above for TrCel45A. TrCel5A-G363A mL column of Biogel P60 equilibrated in 50 mM sodium eluted with Buffer 1 in a substantially pure form as deter citrate, pH 5.0 (Buffer 3). The load was applied at 0.2 mL/min 5 and 4 mL fractions were collected. The load was eluted with mined by SDS-PAGE. TrCel5A-G363 A purified using this two bed volumes of Buffer 3 at 0.2 mL/min. Two major peaks method was tested for activity on carboxymethyl cellulose were present and well separated in the elution profile as using methods known to those skilled in the art and found to determined by absorbance at 280 nm. The second peak was have similar activity (IU/mg protein) as wild-type TrCel5A. TrCel45A as assessed by SDS-PAGE. The identity of this protein was confirmed by mass spectrometry. Fractions cor 10 Example 4 responding to the second peak from the Biogel P60 column were pooled and concentrated as described above. The purity Depilling Activity of Cellulase Mixtures Enriched in of the TrCel45 in the concentrated fractions was confirmed by Family 45 and Family 5 and/or Family 6 Cellulases SDS-PAGE analysis. 15 A blend of the purified TrCel45A and TrCel5A-G363A Example 3.3 cellulases was prepared with a weight ratio of TrCel45: Purification of Trichoderna reesei TrCel5A-G363A TrCel5A of 20:80. The blend of components, as well as the individual purified components, was then assayed for depill A strain of Trichoderma reesei, genetically altered Such ing activity at dosages ranging from 1 to 6 mg total protein per that it secretes the Cel5A (G363A) variant but not wild-type g of fabric. The depilling activity measurements were per TrCel5A (strain P976M, as described in WO2011/ formed as described in Example 1.6. As shown in FIG. 14, the 109905A1), was grown in submerged liquid fermentation 20:80 blend of TrCel45A and TrCel5A releases more fines underconditions that induce cellulase production as knownto from the fabric than either of the purified components in those skilled in the art. The fungal cells were removed from 25 isolation. the fermentation broth by filtration across a glass microfiber A blend of purified TrCel45A, TrCel6A, wild-type filter containing a Harborlite filter bed. TrCel5A and TrCel5A-G363A was prepared having a weight TrCel5A-G363A was then enriched in the cell-free fer ratio of 20:80 TrCel45A:(TrCel6A+TrCel5A+TrCel5A mentation broth by incubating the enzyme mixture under G363A in a ratio of 6:5:5). This blend, as well as the 6:5:5 conditions that substantially precipitate other secreted Tri 30 TrCel6A:TrCel5A:TrCel5A-G363A blend and purified choderma proteins. The cell-free broth was adjusted to 50 TrCel45A, was then assayed for depilling activity at dosages mM sodium citrate, pH 3.5 and incubated at 55° C. for 17 h. ranging from 0.7 to 7 mg total protein per g of fabric. The At the end of this incubation, the enzyme mixture was cen depilling activity measurements were performed as described trifuged at 2000 rpm in a Sorvall Legend RT bench-top cen in Example 1.6. As shown in FIG. 15, the blend containing all trifuge for 5 min. The supernatant, containing TrCel5A 35 three types of cellulases consistently exhibited greater depill G363A was saved for further analysis and purification while ing activity (i.e., fines release) for all dosages than either the the pellet, containing other Trichoderma proteins, was dis TrCel6A+TrCel5A+TrCel5A-G363 blend or the purified carded. TrCel45A.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 115

<21 Oc SEO ID NO 1 <211 LENGTH: 397 <212> TYPE PRT ORGANISM: Trichoderma reesei

<4 OOs SEQUENCE: 1

Gln Gln Thr Val Trp Gly Glin Gly Gly Ile Gly Trp Ser Gly Pro 1. 5 15

Thir Asn. Cys Ala Pro Gly Ser Ala Cys Ser Thr Lieu. Asn Pro 25 3 O

Ala Glin Cys Ile Pro Gly Ala Thr Thir Ilie Thir Thr Ser Thir Arg Pro 35 4 O 45

Pro Ser Gly Pro Thir Th Thir Thir Ala Thir Ser Thir Ser Ser Ser SO 55 60

Thir Pro Pro Thir Ser Ser Gly Wall Arg Phe Ala Gly Wall ASn Ile Ala 65 70

Gly Phe Asp Phe Gly Cys Thir Thr Asp Gly Thir Wall Thir Ser 85 90 95

Wall Tyr Pro Pro Leu Lys ASn Phe Thir Gly Ser Asn Asn Tyr Pro Asp 105 110 US 9.238,806 B2 35 36 - Continued

Gly Ile Gly Glin Met Glin His Phe Wall Asn Asp Asp Gly Met Thir Ile 115 12 O 125

Phe Arg Luell Pro Wall Gly Trp Glin Luell Wall Asn Asn Asn Luell Gly 13 O 135 14 O

Gly Asn Luell Asp Ser Thir Ser Ile Ser Tyr Asp Glin Luell Wall Glin 145 150 155 160

Gly Luell Ser Lell Gly Ala Ile Wall Asp Ile His Asn 1.65 17O 17s

Ala Arg Trp Asn Gly Gly Ile Ile Gly Glin Gly Gly Pro Thir Asn Ala 18O 185 19 O

Glin Phe Thir Ser Lell Trp Ser Glin Luell Ala Ser Tyr Ala Ser Glin 195

Ser Arg Wall Trp Phe Gly Ile Met Asn Glu Pro His Asp Wall Asn Ile 21 O 215 22O

Asn Thir Trp Ala Ala Thir Wall Glin Glu Wall Wall Thir Ala Ile Arg Asn 225 23 O 235 24 O

Ala Gly Ala Thir Ser Glin Phe Ile Ser Luell Pro Gly Asn Asp Trp Glin 245 250 255

Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala Ala Ala Luell Ser Glin 26 O 265 27 O

Wall Thir Asn Pro Asp Gly Ser Thir Thir Asn Luell Ile Phe Asp Wall His 27s 285

Tyr Luell Asp Ser Asp Asn Ser Gly Thir His Ala Glu Thir Thir 29 O 295 3 OO

Asn Asn Ile Asp Gly Ala Phe Ser Pro Lieu Thr Trp Luell Arg Gln 3. OS 310 3 5

Asn Asn Arg Glin Ala Ile Lell Thir Glu Thir Gly Gly Asn Wall Glin 3.25 330 335

Ser Ile Glin Asp Met Glin Glin Ile Lell Asn Glin Asn 34 O 345 35. O

Ser Asp Wall Lell Gly Wall Gly Trp Ala Gly Ser Phe Asp 355 360 365

Ser Thir Wall Lell Thir Glu Thir Pro Thir Ser Gly Asn Ser Trp 37 O 375 38O

Thir Asp Thir Ser Lell Wall Ser Ser Luell Arg 385 390

<210s, SEQ ID NO 2 &211s LENGTH: 397 212. TYPE : PRT &213s ORGANISM: Trichoderma reesei

<4 OOs, SEQUENCE: 2

Glin Glin. Thir Wall Trp Gly Glin Gly Gly Ile Gly Trp Ser Gly Pro 1. 1O 15

Thir Asn Cys Ala Pro Gly Ser Ala Cys Ser Thir Lell Asn Pro 25

Ala Glin Cys Ile Pro Gly Ala Thir Thir Ile Thir Thir Ser Thir Arg Pro 35 4 O 45

Pro Ser Gly Pro Thir Thir Thir Thir Arg Ala Thir Ser Thir Ser Ser Ser SO 55 6 O

Thir Pro Pro Thir Ser Ser Gly Wall Arg Phe Ala Gly Wall Asn Ile Ala 65 70 8O

Gly Phe Asp Phe Gly Thir Thir Asp Gly Thir Wall Thir Ser Lys 85 90 95 US 9.238,806 B2 37 38 - Continued

Wall Pro Pro Lell Asn Phe Thir Gly Ser Asn Asn Tyr Pro Asp 105 11 O

Gly Ile Gly Glin Met Glin His Phe Wall Asn Asp Asp Gly Met Thir Ile 115 12 O 125

Phe Arg Luell Pro Wall Gly Trp Glin Tyr Luell Wall Asn Asn Asn Luell Gly 13 O 135 14 O

Gly Asn Luell Asp Ser Thir Ser Ile Ser Tyr Asp Glin Luell Wall Glin 145 150 155 160

Gly Luell Ser Lell Gly Ala Ile Wall Asp Ile His Asn Tyr 1.65 17O 17s

Ala Arg Trp Asn Gly Gly Ile Ile Gly Glin Gly Gly Pro Thir Asn Ala 18O 185 19 O

Glin Phe Thir Ser Lell Trp Ser Glin Luell Ala Ser Tyr Ala Ser Glin 195

Ser Arg Wall Trp Phe Gly Ile Met Asn Glu Pro His Asp Wall Asn Ile 21 O 215 22O

Asn Thir Trp Ala Ala Thir Wall Glin Glu Wall Wall Thir Ala Ile Arg Asn 225 23 O 235 24 O

Ala Gly Ala Thir Ser Glin Phe Ile Ser Luell Pro Gly Asn Asp Trp Glin 245 250 255

Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala Ala Ala Luell Ser Glin 26 O 265 27 O

Wall Thir Asn Pro Asp Gly Ser Thir Thir Asn Luell Ile Phe Asp Wall His 27s 285

Tyr Luell Asp Ser Asp Asn Ser Gly Thir His Ala Glu Thir Thir 29 O 295 3 OO

Asn Asn Ile Asp Gly Ala Phe Ser Pro Luell Thir Trp Luell Arg Glin 3. OS 310 3 5

Asn Asn Arg Glin Ala Ile Lell Thir Glu Thir Gly Gly Asn Wall Glin 3.25 330 335

Ser Ile Glin Asp Met Glin Glin Ile Lell Asn Glin Asn 34 O 345 35. O

Ser Asp Wall Lell Gly Tyr Wall Gly Trp Ala Gly Ser Phe Asp 355 360 365

Ser Thir Wall Lell Thir Glu Thir Pro Thir Ser Gly Asn Ser Trp 37 O 375 38O

Thir Asp Thir Ser Lell Wall Ser Ser Luell Arg 385 390

<210s, SEQ ID NO 3 &211s LENGTH: 447 212. TYPE : PRT &213s ORGANISM: Trichoderma reesei

<4 OOs, SEQUENCE: 3

Glin Ala Cys Ser Ser Wall Trp Gly Glin Cys Gly Gly Glin Asn Trp Ser 1. 15

Gly Pro Thir Cys Cys Ala Ser Gly Ser Thir Wall Ser Asn Asp 25

Tyr Tyr Ser Glin Cys Lell Pro Gly Ala Ala Ser Ser Ser Ser Ser Thir 35 4 O 45

Arg Ala Ala Ser Thir Thir Ser Arg Wall Ser Pro Thir Thir Ser Arg Ser SO 55 6 O

Ser Ser Ala Thir Pro Pro Pro Gly Ser Thir Thir Thir Arg Wall Pro Pro US 9.238,806 B2 39 40 - Continued

65 70

Wall Gly Ser Gly Thir Ala Thir Ser Gly ASn Pro Phe Wall Gly Wall 85 90 95

Thir Pro Trp Ala Asn Ala Ala Ser Glu Wall Ser Ser Luell Ala 105 11 O

Ile Pro Ser Luell Thir Gly Ala Met Ala Thir Ala Ala Ala Ala Wall Ala 115 12 O 125

Wall Pro Ser Phe Met Trp Luell Asp Thir Luell Asp Thir Pro Luell 13 O 135 14 O

Met Glu Glin Thir Lell Ala Asp Ile Arg Thir Ala Asn Asn Gly Gly 145 150 155 160

Asn Ala Gly Glin Phe Wall Wall Asp Luell Pro Asp Arg Asp 1.65 17O 17s

Ala Ala Luell Ala Ser Asn Gly Glu Tyr Ser Ile Ala Asp Gly Gly Wall 18O 185 19 O

Ala Tyr Asn Ile Asp Thir Ile Arg Glin Ile Wall Wall Glu 195

Ser Asp Ile Arg Thir Lell Luell Wall Ile Glu Pro Asp Ser Luell Ala 21 O 215 22O

Asn Luell Wall Thir Asn Lell Gly Thir Pro Cys Ala Asn Ala Glin Ser 225 23 O 235 24 O

Ala Luell Glu Cys Ile Asn Ala Wall Thir Glin Lell Asn Luell Pro 245 250 255

Asn Wall Ala Met Tyr Lell Asp Ala Gly His Ala Gly Trp Luell Gly Trp 26 O 265 27 O

Pro Ala Asn Glin Asp Pro Ala Ala Glin Luell Phe Ala Asn Wall 27s 285

Asn Ala Ser Ser Pro Arg Ala Luell Arg Gly Luell Ala Thir Asn Wall Ala 29 O 295 3 OO

Asn Asn Gly Trp Asn Ile Thir Ser Pro Pro Ser Thir Glin Gly 3. OS 310 315 32O

Asn Ala Wall Asn Glu Luell Ile His Ala Ile Gly Pro Luell 3.25 330 335

Lell Ala Asn His Gly Trp Ser Asn Ala Phe Phe Ile Thir Asp Glin Gly 34 O 345 35. O

Arg Ser Gly Glin Pro Thir Gly Glin Glin Glin Trp Gly Asp Trp 355 360 365

Asn Wall Ile Gly Thir Gly Phe Gly Ile Arg Pro Ser Ala Asn Thir Gly 37 O 375

Asp Ser Luell Luell Asp Ser Phe Wall Trp Wall Lys Pro Gly Gly Glu Cys 385 390 395 4 OO

Asp Gly Thir Ser Asp Ser Ser Ala Pro Arg Phe Asp Ser His Cys Ala 4 OS 415

Lell Pro Asp Ala Lell Glin Pro Ala Pro Glin Ala Gly Ala Trp Phe Glin 42O 425 43 O

Ala Phe Wall Glin Lell Lell Thir Asn Ala ASn Pro Ser Phe Luell 435 44 O 445

SEQ ID NO 4 LENGTH: 225 TYPE : PRT ORGANISM: Trichoderma reesei

< 4 OOs SEQUENCE: 4. US 9.238,806 B2 41 42 - Continued

Ala Thir Thir Thir Arg Asp Gly Glin Glu Gly Ala Cys 1O 15

Gly Gly Ser Ser Ser Gly Ala Phe Pro Trp Glin Lell Gly Ile Gly 25

Asn Gly Wall Tyr Thir Ala Ala Gly Ser Glin Ala Lell Phe Asp Thir Ala 35 4 O 45

Gly Ala Ser Trp Gly Ala Gly Gly Lys Cys Glin Luell Thir SO 55 6 O

Ser Thir Gly Glin Ala Pro Ser Ser Gly Thir Gly Gly Ala Ala 65 70

Gly Glin Ser Ile Ile Wall Met Wall Thir Asn Luell Pro Asn Asn Gly 85 90 95

Asn Ala Glin Trp Cys Pro Wall Wall Gly Gly Thir Asn Glin Tyr Gly Tyr 105 11 O

Ser His Phe Asp Ile Met Ala Glin Asn Glu Ile Phe Gly Asp Asn 115 12 O 125

Wall Wall Wall Asp Phe Glu Pro Ile Ala Pro Gly Glin Ala Ala Ser 13 O 135 14 O

Asp Trp Gly Thir Lell Wall Gly Glin Glin Glu Thir Asp Pro Thir 145 150 155 160

Pro Wall Luell Gly Asn Asp Thir Gly Ser Thir Pro Pro Gly Ser Ser Pro 1.65 17s

Pro Ala Thir Ser Ser Ser Pro Pro Ser Gly Gly Gly Glin Glin Thir Luell 18O 185 19 O

Gly Cys Gly Gly Ala Gly Trp Thr Gly Pro Thr Thr Gln 195 2OO

Ala Pro Gly Thir Wall Glin Asn Glin Trp Tyr Ser Glin Luell 21 O 215 22O

Pro 225

<210s, SEQ ID NO 5 &211s LENGTH: 497 212. TYPE : PRT &213s ORGANISM: Trichoderma reesei

<4 OOs, SEQUENCE:

Glin Ser Ala Cys Thir Lell Glin Ser Glu Thir His Pro Pro Luell Thir Trp 1. 15

Glin Lys Cys Ser Ser Gly Gly Thir Cys Thir Glin Glin Thir Gly Ser Wall 25 3O

Wall Ile Asp Ala Asn Trp Arg Trp Thir His Ala Thir Asn Ser Ser Thir 35 4 O 45

Asn Cys Asp Gly Asn Thir Trp Ser Ser Thir Lell Pro Asp Asn SO 55 6 O

Glu Thir Ala Asn Luell Asp Gly Ala Ala Ala Ser 65 70

Thir Gly Wall Thir Thir Ser Gly Asn Ser Luell Ser Ile Gly Phe Wall 85 90 95

Thir Glin Ser Ala Glin Asn Wall Gly Ala Arg Lell Tyr Luell Met Ala 1OO 105 11 O

Ser Asp Thir Thir Tyr Glin Glu Phe Thir Luell Luell Gly Asn Glu Phe Ser 115 12 O 125

Phe Asp Wall Asp Wall Ser Glin Luell Pro Gly Lell Asn Gly Ala Luell 13 O 135 14 O US 9.238,806 B2 43 44 - Continued

Tyr Phe Wall Ser Met Asp Ala Asp Gly Wall Ser Lys Tyr Pro Thir 145 150 155 160

Asn Thir Ala Gly Ala Gly Thir Tyr Cys Asp Ser Glin 1.65 17s

Pro Arg Asp Luell Lys Phe Ile Asn Gly Ala Asn Wall Glu Gly Trp 18O 185 19 O

Glu Pro Ser Ser Asn Asn Ala Asn Thir Ile Gly Gly His Gly Ser 195

Cys Ser Glu Met Asp Ile Trp Glu ASn Ser Ile Ser Glu Ala 21 O 215 22O

Lell Thir Pro His Pro Cys Thir Thir Wall Glin Glu Ile Glu Gly 225 23 O 235 24 O

Asp Gly Gly Gly Thir Ser Asp Asn Arg Tyr Gly Gly Thir 245 250 255

Asp Pro Asp Gly Cys Asp Trp Asn Pro Arg Lell Gly Asn Thir Ser 26 O 265 27 O

Phe Gly Pro Gly Ser Ser Phe Thir Luell Asp Thir Thir Luell 28O 285

Thir Wall Wall Thir Glin Phe Glu Thir Ser Gly Ala Ile Asn Arg 29 O 295 3 OO

Wall Glin Asn Gly Wall Thir Phe Glin Glin Pro ASn Ala Glu Luell Gly Ser 3. OS 310 315

Ser Gly Asn Glu Lell Asn Asp Asp Tyr Thir Ala Glu Glu Ala 3.25 330 335

Glu Phe Gly Gly Ser Ser Phe Ser Asp Gly Gly Lell Thir Glin Phe 34 O 345 35. O

Ala Thir Ser Gly Gly Met Wall Luell Wall Met Ser Luell Trp Asp 355 360 365

Asp Tyr Ala Asn Met Lell Trp Luell Asp Ser Thir Pro Thir Asn 37 O 375

Glu Thir Ser Ser Thir Pro Gly Wall Arg Gly Ser Ser Thir Ser 385 390 395 4 OO

Ser Gly Wall Pro Ala Glin Wall Ser Glin Ser Pro Asn Ala Lys Wall 4 OS 415

Thir Phe Ser Asn Ile Phe Pro Ile Gly Ser Thir Gly Asn Pro 425 43 O

Ser Gly Gly Asn Pro Pro Gly Asn Pro Pro Gly Thir Thir Thir Thir 435 445

Arg Arg Pro Ala Thir Thir Thir Ser Ser Pro Gly Pro Thir Glin Ser 450 45.5 460

His Gly Glin Cys Gly Gly Gly Ser Gly Pro Thir Wall 465 470

Ala Ser Gly Thir Thir Glin Wall Luell Asn Pro Tyr Ser Glin 485 490 495

Lell

SEQ ID NO 6 LENGTH: 437 TYPE : PRT ORGANISM: Trichoderma reesei

< 4 OOs SEQUENCE: 6 Gln Glin Pro Gly Thr Ser Thr Pro Glu Val His Pro Llys Lieu. Thir Thr 1. 5 15 US 9.238,806 B2 45 46 - Continued

Thir Lys Ser Gly Gly Cys Wall Ala Glin Asp Thir Ser Wall 2O 25 3O

Wall Luell Asp Trp Asn Arg Trp Met His Asp Ala Asn Asn Ser 35 4 O 45

Thir Wall Asn Gly Gly Wall Asn Thir Thir Luell Cys Pro Asp Glu Ala SO 55 6 O

Thir Gly Asn Cys Phe Ile Glu Gly Wall Asp Ala Ala Ser 65 70

Gly Wall Thir Thir Ser Gly Ser Ser Luell Thir Met Asn Glin Met Pro 85 90 95

Ser Ser Ser Gly Gly Ser Ser Wall Ser Pro Arg Lell Tyr Luell Luell 105 11 O

Asp Ser Asp Gly Glu Wall Met Luell Luell Asn Gly Glin Glu Luell 115 12 O 125

Ser Phe Asp Wall Asp Lell Ser Ala Luell Pro Gly Glu Asn Gly Ser 13 O 135 14 O

Lell Luell Ser Glin Met Asp Glu Asn Gly Gly Ala Asn Asn 145 150 155 160

Thir Ala Gly Ala Asn Gly Ser Gly Tyr Asp Ala Cys Pro 1.65 17O 17s

Wall Glin Thir Trp Arg Asn Gly Thir Luell Asn Thir Ser His Gly Phe 18O 185

Asn Glu Met Asp Ile Luell Glu Gly ASn Ser Arg Asn Ala 195

Lell Thir Pro His Ser Thir Ala Thir Ala Asp Ser Gly 21 O 215 22O

Gly Phe Asn Pro Tyr Gly Ser Gly Ser Pro Gly 225 23 O 235 24 O

Asp Thir Wall Asp Thir Ser Thir Phe Thir Ile Ile Thir Phe Asn 245 250 255

Thir Asp Asn Gly Ser Pro Ser Gly Asn Luell Wall Ser Ile Thir Arg 26 O 265 27 O

Glin Glin Asn Gly Wall Asp Ile Pro Ser Ala Glin Pro Gly Gly Asp 27s 285

Thir Ile Ser Ser Cys Pro Ser Ala Ser Ala Gly Gly Luell Ala Thir 29 O 295 3 OO

Met Gly Ala Lell Ser Ser Gly Met Wall Luell Wall Phe Ser Ile Trp 3. OS 310 315

Asn Asp Asn Ser Glin Met Asn Trp Luell Asp Ser Gly Asn Ala Gly 3.25 330 335

Pro Ser Ser Thir Glu Gly Asn Pro Ser ASn Ile Lell Ala Asn Asn 34 O 345 35. O

Pro Asn Thir His Wall Wall Phe Ser Asn Ile Arg Trp Gly Asp Ile Gly 355 360 365

Ser Thir Thir Asn Ser Thir Ala Pro Pro Pro Pro Pro Ala Ser Ser Thir 37 O 375 38O

Thir Phe Ser Thir Thir Arg Arg Ser Ser Thir Thir Ser Ser Ser Pro Ser 385 390 395 4 OO

Thir Glin Thir His Trp Gly Glin Gly Gly Ile Gly Ser Gly 4 OS 41O 415

Thir Cys Thir Ser Gly Thir Thir Glin Ser Asn Asp 42O 425 43 O US 9.238,806 B2 47 48 - Continued Tyr Ser Glin Cys Lieu 435

<210s, SEQ ID NO 7 &211s LENGTH: 284 212. TYPE: PRT <213> ORGANISM: Humicola insolens

<4 OO > SEQUENCE: 7 Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys Cys Llys Pro Ser Cys 1. 5 1O 15 Gly Trp Ala Lys Lys Ala Pro Val Asn Glin Pro Val Phe Ser Cys Asn 2O 25 3O Ala Asn. Phe Glin Arg Ile Thr Asp Phe Asp Ala Lys Ser Gly Cys Glu 35 4 O 45 Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala Val SO 55 6 O Asn Asp Asp Phe Ala Lieu. Gly Phe Ala Ala Thir Ser Ile Ala Gly Ser 65 70 7s 8O Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu Lieu. Thr Phe Thr Ser 85 90 95 Gly Pro Val Ala Gly Lys Lys Met Val Val Glin Ser Thr Ser Thr Gly 1OO 105 11 O Gly Asp Lieu. Gly Ser Asn His Phe Asp Lieu. Asn. Ile Pro Gly Gly Gly 115 12 O 125 Val Gly Ile Phe Asp Gly Cys Thr Pro Glin Phe Gly Gly Lieu Pro Gly 13 O 135 14 O Glin Arg Tyr Gly Gly Ile Ser Ser Arg Asn. Glu. Cys Asp Arg Phe Pro 145 150 155 160 Asp Ala Lieu Lys Pro Gly Cys Tyr Trp Arg Phe Asp Trp Phe Lys Asn 1.65 17O 17s Ala Asp Asn Pro Ser Phe Ser Phe Arg Glin Val Glin Cys Pro Ala Glu 18O 185 19 O Lieu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp Asp Gly Asn. Phe Pro 195 2OO 2O5

Ala Wall Glin Ile Pro Ser Ser Ser Thir Ser Ser Pro Wall Asn. Glin Pro 21 O 215 22O

Thir Ser Thir Ser Thir Thir Ser Thir Ser Thir Thir Ser Ser Pro Pro Wall 225 23 O 235 24 O Gln Pro Thir Thr Pro Ser Gly Cys Thr Ala Glu Arg Trp Ala Glin Cys 245 250 255 Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys Val Ala Gly Ser Thr 26 O 265 27 O Cys Thir Lys Ile Asn Asp Trp Tyr His Glin Cys Lieu. 27s 28O

<210s, SEQ ID NO 8 &211s LENGTH: 247 212. TYPE: PRT <213> ORGANISM: Trichoderma viride

<4 OOs, SEQUENCE: 8 Met Lys Ala Thr Lieu Val Lieu. Gly Ser Lieu. Ile Val Gly Ala Val Ser 1. 5 1O 15

Ala Tyr Lys Ala Thir Thir Thr Arg Tyr Tyr Asp Gly Glin Glu Gly Ala 2O 25 3O US 9.238,806 B2 49 50 - Continued

Gly Cys Gly Ser Ser Ser Gly Ala Phe Pro Trp Glin Lieu. Gly Ile 35 4 O 45

Gly Asn Gly Wall Tyr Thir Ala Ala Gly Ser Glin Ala Lell Phe Asp Thir SO 55 6 O

Ala Gly Ala Ser Trp Cys Gly Ala Gly Gly Cys Glin Luell 65 70

Thir Ser Thir Gly Glin Ala Pro Ser Ser Gly Thir Gly Gly Ala 85 90 95

Ala Gly Glin Ser Ile Ile Wall Met Wall Thir ASn Lell Pro Asn Asn 105 11 O

Gly Asn Ala Glin Trp Pro Wall Wall Gly Gly Thir Asn Glin Gly 115 12 O 125

Ser His Phe Asp Ile Met Ala Glin ASn Glu Ile Phe Gly Asp 13 O 135 14 O

Asn Wall Wall Wall Asp Phe Glu Pro Ile Ala Cys Pro Gly Glin Ala Ala 145 150 155 160

Ser Asp Trp Gly Thir Lell Wall Gly Glin Glin Glu Thir Asp Pro 1.65 17O 17s

Thir Pro Wall Luell Gly Asn Asp Thir Gly Ser Thir Pro Pro Gly Ser Ser 18O 185 19 O

Pro Pro Ala Thir Ser Ser Ser Pro Pro Ser Gly Gly Gly Glin Glin Thir 195 2OO

Lell Tyr Gly Glin Gly Gly Ala Gly Trp Thir Gly Pro Thir Thir 21 O 215 22O

Gln Ala Pro Gly Thr Cys Wall Gln Asn Gln Trp Ser Gln Cys 225 23 O 235 24 O

Lell Pro Trp Glu Gly Pro Arg 245

<210s, SEQ ID NO 9 &211s LENGTH: 266 212. TYPE : PRT <213> ORGANISM: Penicillium decumbens

<4 OOs, SEQUENCE:

Met Lys Gly Lys Wall Ala Phe Luell Luell Luell Asp Lell Lell Ala Ser Ala 1. 5 1O 15

Ala Ala Tyr Lys Ala Thir Thir Thir Arg Asp Gly Glin Glu Gly 25 3O

Ala Gly Gly Ser Ser Ser Gly Luell Phe Pro Trp Glin Luell Gly 35 4 O 45

Ile Gly Asn Gly Wall Thir Ala Ala Gly Ser Glin Ala Luell Phe Asp SO 55 6 O

Thir Ala Gly Ala Asp Trp Gly Ala Gly Cys Gly Lys 65 70

Lell Thir Ser Thir Gly Glu Pro Pro Lys Asp Gly Thir Gly Gly 85 90 95

Wall Ala Gly Glin Ser Ile Ile Wall Met Wall Thir Asn Lell Cys Pro Tyr 1OO 105 11 O

Asn Gly Asn Glin Glin Trp Pro Asn Pro Gly Ser Thir Asn Glin Tyr 115 12 O 125

Gly Tyr Ser His Phe Asp Ile Met Ala Glin Asn Glu Ile Phe Gly 13 O 135 14 O

Asp Asn Wall Wall Wall Asp Phe Glu Pro Ile Ala Pro Gly Glin Ala 145 150 155 160 US 9.238,806 B2 51 - Continued

Asn Ser Asp Trp Glin Ser Cys Val Cys Tyr Gly Lys Thr Glu. Thir Asp 1.65 17O 17s Thir Thr Pro Val Gly Lieu. Thir Ala Gly Gly Gly Gly Gly Gly Ser Ser 18O 185 19 O Gln Ser Ser Thr Thr Ser Glin Gly Ser Thr Thr Thr Arg Thr Thr Lieu. 195 2OO 2O5 Thr Ala Thr Thr Thr Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 21 O 215 22O Gly Ser Ser Ser Gly Thr Glin Ser Val Tyr Gly Glin Cys Gly Gly Ser 225 23 O 235 24 O Gly Trp Thr Gly Pro Thr Asn Cys Ala Ser Gly Ser Lys Cys Thr Ala 245 250 255 Gln Asn Gln Trp Tyr Ser Glin Cys Lieu Pro 26 O 265

<210s, SEQ ID NO 10 &211s LENGTH: 247 212. TYPE: PRT <213> ORGANISM: Aspergillus nidulans <4 OOs, SEQUENCE: 10 Met Glin Arg Ile Pro Asp Arg Lieu. Ser Asp Ser Thr Arg Ile Thr Arg 1. 5 1O 15 Pro His Arg Ser Lieu. Cys Cys Asn Thr Gly Gly Ala Ser Ser Ser Cys 2O 25 3O Pro Gly Tyr His ASn Cys Ala Cys Gly Cys Gly ASn Lys Ile Gly Thr 35 4 O 45 Tyr Asp Trp Ser Tyr Gly Ile Ala Asn Llys Val Tyr Thr Ala Ala Ala SO 55 6 O Asn Glin Ala Lieu. Phe Asp Ser Gly Pro Asn Asp Ala Thr His Trp Cys 65 70 7s 8O Gly Asin Gly Cys Gly Lys Cys Tyr Arg Lieu. Thir Ser Thr Gly Val Ser 85 90 95 Thr Cys Glu Thr Cys Gly Ala Gly Gly Glu Gln Gly Lys Ser Ile Val 1OO 105 11 O Val Met Val Thr Asn Lieu. Cys Pro Phe Lys Gly Asn Glu Arg Trp Cys 115 12 O 125 Pro Asn Pro Gly Glin Lieu. Asn Pro His Gly Tyr Ala Tyr His Phe Asp 13 O 135 14 O Ile Met Gly Gly Ala Gly Val Phe Gly Asp Asn Val Val Val Glu Phe 145 150 155 160 Glu Glu Val Pro Cys Pro Gly Asp Ala Ala Phe Llys Trp Ala Ala Cys 1.65 17O 17s Glu Cys His Pro Asn Lieu. Arg Asn Lys Asp Lieu. Thir Lieu. Asn Ala Gly 18O 185 19 O

Ala His Ala Ala Gly Ser Lys Ile Val Gly Pro Ala Glin Ala Ala Ile 195 2OO 2O5

Ile Ser Val Asn Gly Lieu Pro Ala Pro Ala Ala Met Ala Val Glin Pro 21 O 215 22O

Pro Pro Pro Pro Pro Pro Pro Ala Pro Ala Arg Pro Ala Ile Glu Ile 225 23 O 235 24 O

Pro Pro Pro Pro Ala Pro Wall 245 US 9.238,806 B2 53 - Continued

<210s, SEQ ID NO 11 &211s LENGTH: 194 212. TYPE: PRT <213> ORGANISM: Halliotis discus discus

<4 OOs, SEQUENCE: 11 Met Lys Thir Ala Val Ser Ile Lieu. Lieu. Lieu. Phe Ala Ala Ser Ala Trp 1. 5 1O 15 Ala Asn Gln Lys Cys Gln Met His Asn Gly Ile Arg Met Tyr Asn Gly 2O 25 3O Llys His Cys Ala Ser Thir Thr Arg Tyr Asn Asp Gly His Lys Gly Ala 35 4 O 45 Cys Gly Cys Gly Glin Asn Asp Thr Pro Phe Pro Trp Asn Asn Asn Glin SO 55 6 O Tyr Val Ala Ala Ala Asn Gln Lys Lieu. Phe Ser Asn. Ser Gly Ser Thr 65 70 7s 8O Trp. Cys Gly Asp Ser Cys Gly Lys Cys Val Lys Lieu. Thir Thr Thr Gly 85 90 95 Gly Ser Ile Pro Gly Ala Gly Thr Gly Ala His Ala Gly Glin Ser His 1OO 105 11 O Val Phe Met Ile Thr Asn Asp Cys Pro Asp Val Ala Pro Asn Lieu. Glu 115 12 O 125 Trp. Cys Ala Gln Lys Gly Ala Pro Gly Ser Gly His Gly Asn Thr His 13 O 135 14 O Gly Tyr Glu Val His Phe Asp Lieu. Glu Asn. Asn Gly Asn Glin Ile Ser 145 150 155 160 Lys Lieu. Gly Trp Asp Asn Pro Glu Val Thir Trp Glu Trp Ser Ser Cys 1.65 17O 17s His Gly Ser Asn Thr Pro Thr Asp Gln Met Trp His Thr Cys Glu. Cys 18O 185 19 O

Ser His

<210s, SEQ ID NO 12 &211s LENGTH: 195 212. TYPE: PRT <213> ORGANISM: Ampullaria crossean

<4 OOs, SEQUENCE: 12 Met Lys Lieu. Phe Tyr Lieu. Lieu. Cys Lieu Ala Val Pro Lieu. Lieu. Glu Ala 1. 5 1O 15 Ala Glin Lieu. Cys Glin Pro Asp Ser Arg Gly Val Arg Arg Phe Asin Gly 2O 25 3O Llys Pro Cys Ala Ser Thir Thr Arg Tyr Val Asp Gly His Lys Gly Ala 35 4 O 45 Cys Gly Cys Gly Gln Lys Gly Ser Asp Thr Pro Phe Pro Trp Asn Ile SO 55 6 O Gln Lys His Val Thr Ala Pro Ser Glu Arg Tyr Phe Asp Gly Gly Gly 65 70 7s 8O

Ser Ser Lieu. Trp Cys Gly Arg Asn. Cys Gly Lys Cys Wall Lys Lieu. Thir 85 90 95 Pro Thr Gly Gly Phe Val Pro Gly Lys Gly Asn Ala Pro Pro Asn His 1OO 105 11 O

Asn Pro Val Val Phe Glin Val Thr Asn Ala Cys Pro Ile Asin Gly Asn 115 12 O 125 Glu Glu Trp Cys Gly Ile Ser Gly Ala Pro Gly Thr Gly His Val Asn 13 O 135 14 O US 9.238,806 B2 55 56 - Continued

Ser His Gly Tyr Glu Val His Phe Asp Luell Glin Asp Glin Val Gly Glin 145 150 155 160

Wall Glu Ala Luell His Trp Asp Asn Pro Glu Wall Thir Trp Glu Glu Thir 1.65 17O

Ser Pro Gly Asp Lieu. Glin Ser Asn Glin Gln Cys Glu. Cys His 18O 185 19 O

Asn Ser Gly 195

<210s, SEQ ID NO 13 &211s LENGTH: 195 212. TYPE : PRT <213> ORGANISM: Ampullaria crossean

<4 OOs, SEQUENCE: 13

Met Lys Lieu Phe Tyr Lell Lell Luell Ala Wall Pro Wall Luell Glu Ala 1. 5 15

Ala Glin Luell Cys Glin Pro Asp Ala His Gly Wall Arg Arg Phe Asn Gly 25

Arg Pro Cys Ala Ser Thir Thir Arg Wall Asp Gly His Gly Ala 35 4 O 45

Gly Gly Glin Gly Ser Asp Thir Pro Phe Pro Trp Asn Luell SO 55 6 O

Glin His Wall Thir Ala Pro Ser Glu Arg Tyr Phe Asp Asp Gly Gly 65 70 7s 8O

Ser Asn Lieu Trp Cys Gly Asn Gly Wall Arg Lieu Thr 85 90 95

Pro Thir Gly Gly Phe Wall Pro Gly Lys Gly Ala Pro Pro Asn His 105 11 O

Asn Pro Wall Wall Phe Met Wall Thir Asn Ala Pro Ile Asn Gly Asn 115 12 O 125

Glu Glu Trp Gly Ile Ser Gly Pro Gly Thir Asn His Wall Asn 13 O 135 14 O

Ser His Gly Glu Wall His Phe Asp Luell Glin Asp Glin Wall Gly Glin 145 150 155 160

Wall Glu Ala Luell His Trp Asp Asn Pro Glu Wall Thir Trp Glu Glu Wall 1.65 17O 17s

Pro Pro Gly Asp Lell Glin Ala Asn Glin Glin Glu His 18O 185 19 O

Asn Ser Asp 195

SEQ ID NO 14 LENGTH: 211 TYPE : PRT ORGANISM: Mytilus edulis

< 4 OOs SEQUENCE: 14

Met Lys Tyr Lieu Val Lell Ser Luell Luell Wall Luell Wall Ser Wall Ser 1. 5 1O 15

Ala Asn Gln Lys Cys Ser Gly Asn Pro Arg Arg Asn Gly Ser 25 3O

Cys Ala Ser Thr Thr Asn His Asp Ser His Gly Ala Gly 35 4 O 45

Cys Gly Pro Ala Ser Gly Asp Ala Glin Phe Gly Trp Asn Ala Gly Ser SO 55 6 O US 9.238,806 B2 57 58 - Continued

Phe Wall Ala Ala Ala Ser Glin Met Tyr Phe Asp Ser Gly Asn Lys Gly 65 70 8O

Trp Gly Glin His Gly Glin Cys Ile Lys Lell Thir Thir Thir Gly 85 90 95

Gly Wall Pro Gly Glin Gly Gly Pro Wall Arg Glu Gly Luell Ser 105 11 O

Thir Phe Met Ile Thir Asn Lell Cys Pro Asn Ile Pro Asn Glin Asp 115 12 O 125

Trp Cys Asn Glin Gly Ser Glin Gly Gly His Asn Gly Tyr 13 O 135 14 O

Glu Luell His Luell Asp Lell Glu Asn Gly Arg Ser Glin Wall Thir Gly Met 145 150 155 160

Gly Trp Asn Asn Pro Glu Thir Thir Trp Glu Wall Wall Asn Asp Ser 1.65 17O 17s

Glu His Asn His Asp His Arg Thir Pro Ser ASn Ser Met Tyr Gly Glin 18O 185 19 O

Glin Cys Ala His Glin Gly Lys Arg Gly Luell Asn Glu Thir Ser Asn 195 2OO 2O5

Glu Ser Luell 21 O

<210s, SEQ ID NO 15 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Phanerochaete chrysosporium

<4 OOs, SEQUENCE: 15

Met Ala Lys Lieu Ser Met Phe Luell Gly Phe Wall Ala Wall Ala Thir Luell 1. 5 15

Ala Ser Ala Luell Thir Wall Ser Glu Lys Arg Ala Thir Gly Gly Tyr Wall 2O 25

Glin Glin Ala Thir Gly Glin Ala Ser Phe Thir Met Ser Gly Gly 35 4 O 45

Ser Pro Ala Gly Ala Ala Ser Gly Phe Thir Ala Ala Ile Asn SO 55 6 O

Glin Luell Ala Phe Gly Ser Ala Pro Gly Luell Gly Ala Gly Asp Ala Cys 65 70

Gly Arg Phe Ala Lell Thir Gly Asn His Asp Pro Ser Pro Asn 85 90 95

Thir Gly Pro Phe Gly Glin Thir Ile Wall Wall Wall Thir Asp Luell 105 11 O

Pro Wall Glin Gly Asn Glin Glu Phe Gly Glin Thir Thir Ser Asn 115 12 O 125

Pro Thir Asn Glin His Gly Met Pro Phe His Phe Asp Ile Glu Asp 13 O 135 14 O

Thir Gly Gly Ser Ala Lys Phe Phe Pro Ser Gly His Gly Ala Luell Thir 145 150 155 160

Gly Thir Phe Thir Glu Wall Ser Ser Glin Trp Ser Gly Ser Asp Gly 1.65 17O 17s

Gly Glin Luell Trp Asn Gly Ala Luell Ser Gly Glu Thir Ala Pro Asn 18O 185 19 O

Trp Pro Ser Thir Ala Gly Asn Gly Thir Ala Pro Ser 195 2OO 2O5 US 9.238,806 B2 59 - Continued

<210s, SEQ ID NO 16 &211s LENGTH: 305 212. TYPE: PRT <213> ORGANISM: Humicola grisea var. thermoidea <4 OOs, SEQUENCE: 16 Met Arg Ser Ser Pro Lieu Lleu Pro Ser Asp Val Val Ala Ala Lieu Pro 1. 5 1O 15 Val Lieu Ala Lieu Ala Ala Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys 2O 25 3O Cys Llys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asin Glin Pro 35 4 O 45 Val Phe Ser Cys Asn Ala Asn. Phe Glin Arg Lieu. Thir Asp Phe Asp Ala SO 55 6 O Llys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Glin 65 70 7s 8O Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Phe Gly Phe Ala Ala Thr 85 90 95 Ser Ile Ala Gly Ser Asn. Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 1OO 105 11 O Lieu. Thir Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Glin 115 12 O 125 Ser Thir Ser Thr Gly Gly Asp Lieu. Gly Ser Asn His Phe Asp Lieu. Asn 13 O 135 14 O Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Glin Phe 145 150 155 160 Gly Gly Lieu Pro Gly Glin Arg Tyr Gly Gly Ile Ser Ser Arg Asn. Glu 1.65 17O 17s Cys Asp Arg Phe Pro Asp Ala Lieu Lys Pro Gly Cys Tyr Trp Arg Phe 18O 185 19 O Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Glin Val 195 2OO 2O5 Glin Cys Pro Ala Glu Lieu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 21 O 215 22O Asp Gly Asn Phe Pro Ala Val Glin Ile Pro Ser Ser Ser Thr Ser Ser 225 23 O 235 24 O Pro Val Gly Glin Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr 245 250 255 Ser Ser Pro Pro Val Glin Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu 26 O 265 27 O Arg Trp Ala Glin Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys 27s 28O 285 Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Glin Cys 29 O 295 3 OO

Lell 3. OS

<210s, SEQ ID NO 17 &211s LENGTH: 306 212. TYPE: PRT <213> ORGANISM: Humicola nigrescens

<4 OOs, SEQUENCE: 17 Pro Phe Met Met Val Ala Trp Trp Ser Leu Phe Lieu. Tyr Gly Lieu. Glin 1. 5 1O 15

Val Ala Ala Pro Ala Phe Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trip US 9.238,806 B2 61 62 - Continued

25

Asp Cys Pro Ser Ser Trp Pro Gly Ala Luell Wall Asn 35 4 O 45

Glin Pro Wall Ala Arg Asn Ala Asn Phe Glin Arg Ile Thir Asp Pro SO 55 6 O

Asn Ala Ser Gly Cys Asp Gly Gly Ser Ala Phe Ser Ala Asp 65 70

Glin Thir Pro Trp Ala Wall Ser Asp Asp Phe Ala Gly Phe Ala Ala 85 90 95

Thir Ala Luell Ala Gly Glin Ser Glu Ser Ser Trp Ala 105 11 O

Glu Luell Thir Phe Thir Ser Gly Pro Wall Ala Gly Lys Met Ala Wall 115 12 O 125

Glin Ser Thir Ser Thir Gly Gly Asp Luell Gly Ser Asn His Phe Asp Luell 13 O 135 14 O

Asn Met Pro Gly Gly Gly Wall Gly Ile Phe Asp Gly Ser Pro Glin 145 150 155 160

Wall Gly Gly Luell Ala Gly Glin Arg Gly Gly Wall Ser Ser Arg Ser 1.65 17O

Glu Asp Ser Phe Pro Ala Ala Luell Pro Gly Tyr Trp Arg 18O 185 19 O

Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Glin 195

Wall Glin Pro Ala Glu Lell Wall Ala Arg Thir Gly Arg Arg Asn 210 215 220

Asp Asp Gly Asn Phe Pro Ala Wall Glin Ile Pro Ser Ser Ser Thir Ser 225 23 O 235 24 O

Ser Pro Wall Asn Glin Pro Thir Ser Thir Ser Thir Thir Ser Thir Ser Thir 245 250 255

Thir Ser Ser Pro Pro Wall Glin Pro Thir Thir Pro Ser Gly Cys Thir Ala 26 O 265 27 O

Glu Arg Trp Ala Glin Gly Gly Asn Gly Trp Ser Gly Thir Thir 285

Wall Ala Gly Ser Thir Cys Thir Ile ASn Asp Trp His Glin 29 O 295 3 OO

Cys Luell 3. OS

<210s, SEQ ID NO 18 &211s LENGTH: 316 212. TYPE : PRT &213s ORGANISM: Staphylotrichum coccosporum

<4 OOs, SEQUENCE: 18

Met Arg Ser Ser Pro Wall Lell Arg Thir Ala Luell Ala Ala Ala Luell Pro 1. 5 15

Lell Ala Ala Luell Ala Ala Asp Gly Lys Ser Thir Arg Trp Asp 2O 25

Pro Ser Cys Ser Trp Pro Gly Lys Ala Ser Wall Asn Glin Pro 35 4 O 45

Wall Phe Ala Ser Ala Asn Phe Glin Arg Ile Ser Asp Pro Asn Wall SO 55 6 O

Lys Ser Gly Asp Gly Gly Ser Ala Tyr Ala Ala Asp Glin Thir 65 70 7s 8O US 9.238,806 B2 63 64 - Continued

Pro Trp Ala Wall Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thir Ser 85 90 95

Ile Ser Gly Gly Asn Glu Ala Ser Trp Gly Cys Tyr Glu Luell 105 11 O

Thir Phe Thir Ser Gly Pro Wall Ala Gly Thir Met Wall Wall Glin Ser 115 12 O 125

Thir Ser Thir Gly Gly Asp Lell Gly Thir Asn His Phe Asp Luell Ala Met 13 O 135 14 O

Pro Gly Gly Gly Wall Gly Ile Phe Asp Gly Cys Ser Pro Glin Phe Gly 145 150 155 160

Gly Luell Ala Gly Asp Arg Gly Gly Wall Ser Ser Arg Ser Glin Cys 1.65 17O 17s

Asp Ser Phe Pro Ala Ala Lell Pro Gly Trp Arg Phe Asp 18O 185 19 O

Trp Phe Lys Asn Ala Asp Asn Pro Thir Phe Thir Phe Arg Glin Wall Glin 195 2OO

Pro Ser Glu Lell Wall Ala Arg Thir Gly Arg Arg Asn Asp Asp 21 O 215

Gly Asn Phe Pro Wall Phe Thir Pro Pro Ser Gly Gly Glin Ser Ser Ser 225 23 O 235 24 O

Ser Ser Ser Ser Ser Ser Ala Pro Thir Ser Thir Ser Thir Ser Thir 245 250 255

Thir Ser Thir Lys Ala Thir Ser Thir Thir Ser Thir Ala Ser Ser Glin Thir 26 O 265 27 O

Ser Ser Ser Thr Gly Gly Gly Cys Ala Ala Gln Arg Trp Ala Gln Cys 27s 28O 285

Gly Gly Ile Gly Phe Ser Gly Thir Thir Wall Ser Gly Thir Thir 29 O 295 3 OO

Cys Asn Glin Asn Asp Trp Ser Glin Cys Lell 3. OS 310 315

<210s, SEQ ID NO 19 &211s LENGTH: 235 212. TYPE : PRT &213s ORGANISM: Melanocarpus albomyces

<4 OOs, SEQUENCE: 19

Met Arg Ser Thr Pro Wall Lell Arg Ala Luell Luell Ala Ala Ala Luell Pro 1. 5 1O 15

Lell Gly Ala Luell Ala Ala Asn Gly Glin Ser Thir Arg Trp Asp Cys 2O 25

Lys Pro Ser Cys Gly Trp Arg Gly Gly Pro Wall Asn Glin Pro 35 4 O 45

Wall Tyr Ser Asp Ala Asn Phe Glin Arg Ile His Asp Phe Asp Ala SO 55 6 O

Wall Ser Gly Glu Gly Gly Pro Ala Phe Ser Ala Asp His Ser 65 70 7s

Pro Trp Ala Ile Asn Asp Asn Luell Ser Tyr Gly Phe Ala Ala Thir Ala 85 90 95

Lell Ser Gly Glin Thir Glu Glu Ser Trp Ala Tyr Ala Luell 1OO 105 11 O

Thir Phe Thir Ser Gly Pro Wall Ala Gly Thir Met Wall Wall Glin Ser 115 12 O 125

Thir Ser Thir Gly Gly Asp Lell Gly Ser Asn His Phe Asp Luell Asn Ile 13 O 135 14 O US 9.238,806 B2 65 66 - Continued

Pro Gly Gly Gly Wall Gly Lell Phe Asp Gly Cys Thir Pro Glin Phe Gly 145 150 155 160

Gly Luell Pro Gly Ala Arg Gly Gly Ile Ser Ser Arg Glin Glu 1.65 17O 17s

Asp Ser Phe Pro Glu Pro Lell Pro Gly Cys Glin Trp Arg Phe Asp 18O 185 19 O

Trp Phe Glin Asn Ala Asp Asn Pro Ser Phe Thir Phe Glu Arg Wall Glin 195 2OO

Pro Glu Glu Lell Wall Ala Arg Thir Gly Cys Arg Arg His Asp Asp 21 O 215 22O

Gly Gly Phe Ala Wall Phe Ala Pro Ser Ala 225 23 O 235

<210s, SEQ ID NO 2 O &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Podospora anserina

<4 OOs, SEQUENCE:

Met Arg Ser Ser Thir Wall Lell Glin Thir Ser Luell Lell Ala Wall Luell Pro 1. 5 15

Lell Ala Wall Glin Ala Glin Gly Ala Ser Gly Ser Gly Ser Thir Arg 25 3O

Trp Asp Cys Pro Ser Ala Trp Pro Gly Ala Ala 35 4 O 45

Wall Asn Arg Pro Wall Phe Ala Asp Ala Asn Phe Gln Ile Ser SO 55 6 O

Asp Ser Gly Wall Ala Ser Gly Asn Gly Gly Ser Ala Ser Cys 65 70

Ala Asp His Ser Ala Trp Ala Ile Asn Asp ASn Lell Ser Gly Phe 85 90 95

Ala Ala Thir Ala Lell Ser Gly Gly Ser Glu Ala Ser Trp Cys Ala 105 11 O

Glu Luell Thir Phe Thir Asp Gly Pro Wall Ala Gly Lys Met 115 12 O 125

Wall Wall Glin Ser Thir Ser Thir Gly Gly Asp Luell Gly Ser Asn His Phe 13 O 135 14 O

Asp Luell Asn Ile Pro Gly Gly Gly Wall Gly Luell Phe Asp Gly Lys 145 150 155 160

Pro Glin Phe Gly Gly Lell Pro Gly Ala Thir Gly Gly Ile Ser Asp 1.65 17O 17s

Arg Ser Glin Cys Ala Ser Phe Pro Asp Ala Luell Pro Gly Asn 18O 185 19 O

Trp Arg Phe Asp Trp Phe Asn Ala Asp ASn Pro Ser Phe Thir Phe 195

Arg Glin Wall Glin Pro Ser Glu Luell Thir Ala Arg Ser Gly 21 O 215

Arg Asp Asp Asp Ser Arg Phe Pro Wall Phe Ser Pro Pro Gly Gly Gly 225 23 O 235 24 O

Ser Glin Pro Glin Pro Glin Pro Thir Ser Ser Ala Ala Glin Asn Pro Asn 245 250 255

Pro Thir Pro Ser Ala Ala Pro Gly Gly Cys Arg Ala Ala Lys Ala 26 O 265 27 O

Glin Gly Gly Glin Gly Phe Thir Gly Thir Thir Glu Ala Gly US 9.238,806 B2 67 - Continued

27s 28O 285 Ser Thr Cys Thr Ala Ser Asn Gln Trp Tyr Ser Glin Cys Lieu. 29 O 295 3 OO

<210s, SEQ ID NO 21 &211s LENGTH: 315 212. TYPE: PRT <213> ORGANISM: Acremonium thermophilum

<4 OOs, SEQUENCE: 21 Met Arg Ser Ser Pro Phe Lieu. Arg Ala Ala Lieu Ala Ala Ala Lieu Pro 1. 5 1O 15 Lieu. Ser Ala His Ala Lieu. Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys 2O 25 3O Cys Llys Pro Ser Cys Gly Trp Ala Gly Lys Ala Ser Val Asin Glin Pro 35 4 O 45 Val Phe Ser Cys Ser Ala Asp Trp Glin Arg Ile Ser Asp Phe Asn Ala SO 55 6 O Llys Ser Gly Cys Asp Gly Gly Ser Ala Tyr Ser Cys Ala Asp Glin Thr 65 70 7s 8O Pro Trp Ala Val Asn Asp Asn. Phe Ser Tyr Gly Phe Ala Ala Thr Ala 85 90 95 Ile Ala Gly Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Lieu. 1OO 105 11 O Thr Phe Asin Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Glin Ser 115 12 O 125 Thir Ser Thr Gly Gly Asp Lieu. Gly Ser Asn Glin Phe Asp Lieu Ala Ile 13 O 135 14 O Pro Gly Gly Gly Val Gly Ile Phe Asin Gly Cys Ala Ser Glin Phe Gly 145 150 155 160 Gly Lieu Pro Gly Ala Glin Tyr Gly Gly Ile Ser Asp Arg Ser Glin Cys 1.65 17O 17s Ser Ser Phe Pro Ala Pro Leu Gln Pro Gly Cys Gln Trp Arg Phe Asp 18O 185 19 O Trp Phe Glin Asn Ala Asp Asn Pro Thr Phe Thr Phe Glin Arg Val Glin 195 2OO 2O5 Cys Pro Ser Glu Lieu. Thir Ser Arg Thr Gly Cys Lys Arg Asp Asp Asp 21 O 215 22O Ala Ser Tyr Pro Val Phe Asn Pro Pro Ser Val Pro Gly Lieu. Asp Gly 225 23 O 235 24 O Ser Asn Pro Gly Asn Pro Thr Thr Thr Val Val Pro Pro Ala Ser Thr 245 250 255 Ser Thr Ser Arg Pro Thir Ser Ser Thr Ser Ser Pro Val Ser Thr Pro 26 O 265 27 O Thr Gly Glin Pro Gly Gly Cys Thr Thr Glin Llys Trp Gly Glin Cys Gly 27s 28O 285

Gly Ile Gly Tyr Thr Gly Cys Thr Asn Cys Val Ala Gly Thr Thr Cys 29 O 295 3 OO

Thr Glin Lieu. Asn Pro Trp Tyr Ser Glin Cys Lieu. 3. OS 310 315

<210s, SEQ ID NO 22 &211s LENGTH: 299 212. TYPE: PRT <213> ORGANISM: Thiellavia terrestris US 9.238,806 B2 69 70 - Continued

<4 OOs, SEQUENCE: 22

Met Arg Ser Thr Pro Wall Lell Arg Thir Thir Luell Ala Ala Ala Luell Pro 1. 15

Lell Wall Ala Ser Ala Ala Ser Gly Ser Gly Glin Ser Thir Arg Trp 2O 25 3O

Asp Cys Pro Ser Ala Trp Pro Gly Ala Ala Wall Ser 35 4 O 45

Glin Pro Wall Ala Asp Ala Asn Phe Glin Lell Ser Asp Phe SO 55

Asn Wall Glin Ser Gly Cys Asn Gly Gly Ser Ala Ser Ala Asp 65 70

Glin Thir Pro Trp Ala Wall Asn Asp Asn Luell Ala Gly Phe Ala Ala 85 90 95

Thir Ser Ile Ala Gly Gly Ser Glu Ser Ser Trp Ala 105 11 O

Ala Luell Thir Phe Thir Ser Gly Pro Wall Ala Gly Thir Met Wall Wall 115 12 O 125

Glin Ser Thir Ser Thir Gly Gly Asp Luell Gly Ser Asn Glin Phe Asp Ile 13 O 135 14 O

Ala Met Pro Gly Gly Gly Wall Gly Ile Phe ASn Gly Ser Ser Glin 145 150 155 160

Phe Gly Gly Luell Pro Gly Ala Glin Gly Gly Ile Ser Ser Arg Asp 1.65 17O

Glin Asp Ser Phe Pro Ala Pro Luell Pro Gly Glin Trp Arg 18O 185 19 O

Phe Asp Trp Phe Glin Asn Ala Asp Asn Pro Thir Phe Thir Phe Glin Glin 195

Wall Glin Pro Ala Glu Ile Wall Ala Arg Ser Gly Arg Asn 21 O 215 22O

Asp Asp Ser Ser Phe Pro Wall Phe Thir Pro Pro Ser Gly Gly Asn Gly 225 23 O 235 24 O

Gly Thir Gly Thir Pro Thir Ser Thir Ala Pro Gly Ser Gly Glin Thir Ser 245 250 255

Pro Gly Gly Gly Ser Gly Thir Ser Glin Trp Ala Glin Gly 26 O 265 27 O

Gly Ile Gly Phe Ser Gly Thir Thir Cys Wall Ser Gly Thir Thir 285

Glin Lys Luell Asn Asp Tyr Ser Glin Cys Luell 29 O 295

SEQ ID NO 23 LENGTH: 3. Of TYPE : PRT ORGANISM: Trichothecium roseum

< 4 OOs SEQUENCE: 23

Pro Phe Met Met Wall Ala Trp Trp Ser Luell Phe Lell Gly Luell Glin 1. 5 1O 15

Wall Ala Ala Pro Ala Phe Ala Ala Asp Gly Arg Ser Thir Arg Trp 25 3O

Asp Cys Cys Llys Pro Ser Ser Trp Pro Asp Ala Pro Wall Gly 35 4 O 45

Ser Pro Val Gly Thir Asp Ala Gly Asn Ser Pro Lell Gly Asp Pro SO 55 6 O US 9.238,806 B2 71 72 - Continued

Lell Ala Ser Gly Cys Glu Gly Gly Pro Ser Tyr Thir Cys Ala Asn 65 70

Glin Pro Trp Ala Wall Asn Asp Glin Luell Ala Gly Phe Ala Ala 85 90 95

Thir Ala Ile Asn Gly Gly Thir Glu Asp Ser Trp Ala Tyr 105 11 O

Luell Thir Phe Thir Asp Gly Pro Ala Ser Gly Thir Met Ile Wall 115 12 O 125

Glin Ser Thir Asn Thir Gly Gly Asp Luell Ser Asp Asn His Phe Asp Luell 13 O 135 14 O

Lell Ile Pro Gly Gly Gly Wall Gly Ile Phe Asp Gly Thir Ser Glin 145 150 155 160

Gly Glin Ala Lell Pro Gly Ala Glin Tyr Gly Gly Wall Ser Ser Arg 1.65 17O 17s

Ala Glu Asp Glin Met Pro Glu Ala Ile Ala Gly Cys Glin Trp 18O 185 19 O

Arg Asp Trp Phe Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg 195

Glin Wall Glin Pro Ala Glu Luell Wall Ala Arg Thir Gly Arg Arg 21 O 215

Asn Asp Asp Gly Asn Phe Pro Ala Wall Glin Ile Pro Ser Ser Ser Thir 225 23 O 235 24 O

Ser Ser Pro Wall Asn Glin Pro Thir Ser Thir Ser Thir Thir Ser Thir Ser 245 250 255

Thr Thr Ser Ser Pro Pro Wall Gln Pro Thr Thr Pro Ser Gly Cys Thr 26 O 265 27 O

Ala Glu Arg Trp Ala Glin Gly Gly Asn Gly Trp Ser Gly Thir 28O 285

Thir Cys Wall Ala Gly Ser Thir Thir Ile Asn Asp Trp His 29 O 295 3 OO

Glin Luell 3. OS

<210s, SEQ ID NO 24 &211s LENGTH: 297 212. TYPE : PRT <213> ORGANISM: Acremonium thermophilum

<4 OOs, SEQUENCE: 24

Met Arg Ser Ser Pro Phe Lell Arg Ala Ala Luell Ala Ala Ala Luell Pro 1. 5 1O 15

Lell Ser Ala His Ala Lell Asp Gly Lys Ser Thir Arg Trp Asp 2O 25 3O

Pro Ser Gly Trp Pro Gly Ala Ser Wall Asn Glin Pro 35 4 O 45

Wall Phe Ser Ser Ala Asp Trp Glin Arg Ile Ser Asp Phe Asn Ala SO 55 6 O

Lys Ser Gly Asp Gly Gly Ser Ala Tyr Ser Ala Asp Glin Thir 65 70 7s

Pro Trp Ala Wall Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thir Ala 85 90 95

Ile Ala Gly Gly Ser Glu Ser Ser Trp Cys Ala Tyr Ala Luell 1OO 105 11 O

Thir Phe Asn Ser Gly Pro Wall Ala Gly Thir Met Wall Wall Glin Ser 115 12 O 125 US 9.238,806 B2 73 74 - Continued

Thir Ser Thir Gly Gly Asp Lell Gly Ser Asn Glin Phe Asp Luell Ala Ile 13 O 135 14 O

Pro Gly Gly Gly Wall Gly Ile Phe Asn Gly Cys Ala Ser Glin Phe Gly 145 150 155 160

Gly Luell Pro Gly Ala Glin Tyr Gly Gly Ile Ser Asp Arg Ser Glin 1.65 17O 17s

Ser Ser Phe Pro Ala Pro Lell Glin Pro Gly Cys Glin Trp Arg Phe Asp 18O 185 19 O

Trp Phe Glin Asn Ala Asp Asn Pro Thir Phe Thir Phe Glin Arg Wall Glin 195

Pro Ser Glu Lell Thir Ser Arg Thir Gly Cys Lys Arg Asp Asp Asp 21 O 215 22O

Ala Ser Pro Wall Phe Asn Pro Pro Ser Gly Gly Ser Pro Ser Thir 225 23 O 235 24 O

Thir Ser Thir Thir Thir Ser Ser Pro Ser Gly Pro Thir Gly Asn Pro Pro 245 250 255

Gly Gly Gly Gly Cys Thir Ala Glin Lys Trp Ala Glin Gly Gly Thir 26 O 265 27 O

Gly Phe Thir Gly Thir Thir Cys Wall Ser Gly Thir Thir Glin Wall 27s 28O 285

Glin Asn Glin Trp Tyr Ser Glin Luell 29 O 295

SEO ID NO 25 LENGTH: TYPE : PRT ORGANISM: Fusarium anguioides

< 4 OOs SEQUENCE: 25

Pro Phe Met Met Wall Ala Trp Trp Ser Luell Phe Lell Tyr Gly Luell Glin 1. 5 15

Wall Ala Ala Pro Ala Phe Ala Ala Asp Gly Arg Ser Thir Arg Trp 25

Asp Cys Pro Ser Ser Trp Gly Gly Ala Ala Wall Ser 35 4 O 45

Ala Pro Ala Luell Thir Asp Asp ASn Pro Ile Ser Asn Luell SO 55 6 O

Asn Ala Wall Asn Gly Cys Glu Gly Gly Gly Ser Ala Phe Ala Thir 65 70

Asn Tyr Ser Pro Trp Ala Wall Asn Asp Asn Luell Ala Gly Phe Ala 85 90 95

Ala Thir Luell Ala Gly Gly Ser Glu Gly Ser Trp Cys Ala 105 11 O

Ala Luell Thir Phe Thir Thir Gly Pro Wall Gly Lys Thir Met Wall 115 12 O 125

Wall Glin Ser Thir Asn Thir Gly Gly Asp Luell Gly Asp Asn His Phe Asp 13 O 135 14 O

Lell Met Met Pro Gly Gly Gly Wall Gly Ile Phe Asp Gly Thir Ser 145 150 155 160

Glin Phe Gly Ala Lell Gly Gly Ala Glin Tyr Gly Gly Ile Ser Ser 1.65 17O 17s

Arg Ser Glu Cys Asp Ser Phe Pro Glu Thir Luell Asp Gly Cys His 18O 185 19 O

Trp Arg Phe Asp Trp Phe Asn Ala Asp ASn Pro Ser Phe Ser Phe US 9.238,806 B2 75 76 - Continued

195 2OO 2O5

Arg Glin Wall Glin Cys Pro Ala Glu Luell Wall Ala Arg Thir Gly Arg 21 O 215

Arg Asn Asp Asp Gly Asn Phe Pro Ala Wall Glin Ile Pro Ser Ser Ser 225 23 O 235 24 O

Thir Ser Ser Pro Wall Asn Glin Pro Thir Ser Thir Ser Thir Thir Ser Thir 245 250 255

Ser Thir Thir Ser Ser Pro Pro Wall Glin Pro Thir Thir Pro Ser Gly 26 O 265 27 O

Thir Ala Glu Arg Trp Ala Glin Cys Gly Gly ASn Gly Trp Ser Gly 27s 285

Thir Thir Wall Ala Gly Ser Thir Thir Lys Ile Asn Asp Trp 29 O 295 3 OO

His Glin Luell 3. OS

<210s, SEQ ID NO 26 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Clonostachys rosea f. catenulata

<4 OOs, SEQUENCE: 26

Met Met Wall Ala Trp Trp Ser Luell Phe Luell Tyr Gly Lell Glin Wall Ala 1. 5 1O 15

Ala Pro Ala Phe Ala Ala Asp Gly Arg Ser Thir Arg Trp Asp 2O 25

Pro Ser Cys Ala Trp Ser Gly Ala Ser Wall Ser Ser Pro 35 4 O 45

Wall Arg Thir Asp Ala Asn Asn Ser Pro Luell Ser Asp Wall Asp Ala SO 55 6 O

Lys Ser Ala Asp Gly Gly Wall Ala Thir Ser Asn Asn Ala 65 70

Pro Trp Ala Wall Asn Asp Asn Luell Ser Gly Phe Ala Ala Thir Ala 85 95

Ile Asn Gly Gly Ser Glu Ser Ser Trp Ala Tyr Luell 105 11 O

Thir Phe Thir Ser Gly Pro Ala Ser Gly Wall Met Wall Wall Glin Ser 115 12 O 125

Thir Asn Thir Gly Tyr Asp Lell Ser Asn Asn His Phe Asp Ile Luell Met 13 O 135 14 O

Pro Gly Gly Gly Wall Gly Ala Phe Asp Gly Cys Ser Arg Glin Gly 145 150 155 160

Ser Ile Pro Gly Glu Arg Gly Gly Wall Thir Ser Arg Asp Glin 1.65 17O 17s

Asp Glin Met Pro Ser Ala Lell Glin Gly Trp Arg Phe Asp 18O 185 19 O

Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Glin Wall Glin 195 2OO

Pro Ala Glu Lell Wall Ala Arg Thir Gly Arg Arg Asn Asp Asp 21 O 215 22O

Gly Asn Phe Pro Ala Wall Glin Ile Pro Ser Ser Ser Thir Ser Ser Pro 225 23 O 235 24 O

Wall Asn Glin Pro Thir Ser Thir Ser Thir Thir Ser Thir Ser Thir Thir Ser 245 250 255 US 9.238,806 B2 77 - Continued

Ser Pro Pro Val Glin Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu Arg 26 O 265 27 O Trp Ala Glin Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys Val 27s 28O 285 Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Glin Cys Lieu. 29 O 295 3 OO

<210s, SEQ ID NO 27 &211s LENGTH: 293 212. TYPE: PRT <213> ORGANISM: Neurospora crassa

<4 OOs, SEQUENCE: 27 Met Arg Ser Ser Thr Ile Leu Gln Thr Gly Lieu Val Ala Ala Leu Pro 1. 5 1O 15 Phe Ala Val Glin Ala Ala Ser Gly Ser Gly Glin Ser Thr Arg Tyr Trp 2O 25 3O Asp Cys Cys Llys Pro Ser Cys Ser Trp Ser Gly Lys Ala Pro Val Asn 35 4 O 45 Arg Pro Val Lieu Ala Cys Asp Ala Asn. Asn. Asn Pro Lieu. Ser Asp Ala SO 55 6 O Ser Val Lys Ser Gly Cys Asp Gly Gly Ser Ala Tyr Thr Cys Ala Asn 65 70 7s 8O Asn Ser Pro Trp Ala Val Asn Asp Gln Lieu. Ser Tyr Gly Phe Ala Ala 85 90 95 Thr Lys Lieu Ser Gly Gly Thr Glu Ser Ser Trp Cys Cys Ala Cys Tyr 1OO 105 11 O Ala Lieu. Thir Phe Thr Ser Gly Pro Val Ala Gly Lys Thr Lieu Val Val 115 12 O 125 Gln Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Ile 13 O 135 14 O Asn Met Pro Gly Gly Gly Val Gly Lieu. Phe Asp Gly Cys Lys Arg Glin 145 150 155 160 Phe Gly Gly Lieu Pro Gly Ala Glin Tyr Gly Gly Ile Ser Ser Arg Ser 1.65 17O 17s Glin Cys Asp Ser Phe Pro Ala Ala Lieu Lys Pro Gly Cys Glin Trp Arg 18O 185 19 O Phe Asp Trp Phe Glin Asn Ala Asp Asn Pro Asn Phe Thr Phe Lys Glin 195 2OO 2O5 Val Glin Cys Pro Ser Glu Lieu. Thir Ser Arg Thr Gly Cys Lys Arg Asn 21 O 215 22O Asp Asp Ser Glin Phe Pro Val Phe Thr Pro Pro Ser Gly Gly Gly Ser 225 23 O 235 24 O Asn Pro Ser Thr Pro Thr Thr Pro Pro Ser Ser Gly Gly Gly Ser Gly 245 250 255 Cys Thr Ala Asp Llys Tyr Ala Glin Cys Gly Gly Ser Gly Trp Ser Gly 26 O 265 27 O

Cys Thr Asn Cys Pro Ser Gly Ser Thr Cys Llys Thr Ile Asin Asp Tyr 27s 28O 285 Tyr His Glin Cys Ala 29 O

<210s, SEQ ID NO 28 &211s LENGTH: 310 212. TYPE: PRT <213> ORGANISM: Wolutella colletotrichoides US 9.238,806 B2 79 80 - Continued

<4 OOs, SEQUENCE: 28

Met Arg Ser Ser Ala Val Lieu. Ile Gly Luell Wall Ala Gly Wall Ala Ala 1. 5 1O 15

Gln Ser Ser Gly Thr Gly Arg Thr Thir Arg Trp Asp Cys Cys Lys 2O 25 3O

Pro Ser Cys Gly Trp Asp Glu Lys Ala Ser Wall Ser Glin Pro Wall Lys 35 4 O 45

Thir Cys Asp Arg Asn. Asn. Asn Pro Luell Ala Ser Thir Ala Arg Ser Gly SO 55 6 O

Cys Asp Ser Asn Gly Val Ala Tyr Thir ASn Asp Asn Gln Pro Trp 65 70

Ala Val Asn Asp Asn Lieu Ala Tyr Gly Phe Ala Ala Thir Ala Phe Ser 85 90 95

Gly Gly Ser Glu Ala Ser Trp Cys Cys Ala Ala Lieu. Glin Phe 1OO 105 11 O

Thir Ser Gly Pro Val Ala Gly Lys Thir Met Wall Wall Glin Ser Thr Asn 115 12 O 125

Thr Gly Gly Asp Lieu. Ser Gly Asn His Phe Asp Ile Lell Met Pro Gly 13 O 135 14 O

Gly Gly Lieu. Gly Ile Phe Asp Gly Thir Pro Glin Trp Gly Val Ser 145 150 155 160

Phe Pro Gly Asn Arg Tyr Gly Gly Thir Thir Ser Arg Ser Gln Cys Ser 1.65 17O 17s

Glin Ile Pro Ser Ala Leu Gln Pro Gly Asn Trp Arg Tyr Asp Trp 18O 185 19 O

Phe Asn Asp Ala Asp ASn Pro Asp Wall Ser Trp Arg Arg Wall Glin Cys 195 2OO

Pro Ala Ala Lieu. Thir Asp Arg Thr Gly Arg Arg Ser Asp Asp Gly 21 O 215

Asn Tyr Pro Val Phe Glin Pro Gly Pro Pro Pro Ala Thir Thir Ile Arg 225 23 O 235 24 O

Thir Ser Thir Thir Ile Thir Ala Ser Ser Ser Ser Ser Ser Ser Ser Ser 245 250 255

Ser Thir Thr Ala Gly Ser Pro Pro Wall Pro Thir Gly Gly Gly Ser Gly 26 O 265 27 O

Pro Thir Ser Pro Val Trp Gly Glin Gly Gly Glin Gly Trp Ser Gly 27s 28O 285

Pro Thr Arg Cys Val Ala Gly Ser Thir Cys Ser Wall Wall As Pro Trp 29 O 295 3 OO Tyr Ser Glin Cys Phe Pro 3. OS 310

<210s, SEQ ID NO 29 &211s LENGTH: 355 212. TYPE: PRT <213> ORGANISM: Gibberella zeae

<4 OOs, SEQUENCE: 29

Met Arg Ser Phe Ala Lieu. Lieu Ala Leu Phe Gly Pro Lell Ala Wall Ser 1. 5 15

Ala Ala Ser Gly Ser Gly His Ser Thr Arg Tyr Trp Asp Cys Cys Lys 2O 25 3O

Pro Ser Cys Ser Trp Ser Gly Lys Ala Lys Val Ser Ala Pro Ala Luell 35 4 O 45 US 9.238,806 B2 81 82 - Continued

Thir Cys Asp Lys Asp Asn Pro Ile Thir ASn Lell Asn Ala Wall Asn SO 55 6 O

Gly Glu Ser Gly Gly Ser Ala Phe Ala Cys Thir Asn Ser Pro 65 70

Trp Ala Wall Asn Asp Asp Lell Ala Gly Phe Thir Ala Thir Lys Luell 85 90 95

Ala Gly Gly Thir Glu Ala Ser Trp Cys Ala Ala Luell Thir 105 11 O

Phe Thir Thir Gly Pro Wall Gly Lys Met Ile Wall Glin Ser Thir 115 12 O 125

Asn Thir Gly Gly Asp Lell Gly Asp Asn His Phe Asp Lell Met Met Pro 13 O 135 14 O

Gly Gly Gly Wall Gly Ile Phe Asp Gly Thir Ser Glu Phe Gly Lys 145 150 155 160

Pro Luell Gly Gly Ala Glin Tyr Gly Gly Ile Ser Ser Arg Ser Glin 1.65 17O 17s

Asp Ser Phe Pro Glu Lell Lell Asp Gly Cys His Trp Arg Phe Asp 18O 185 19 O

Trp Phe Lys Asn Ala Asp Asn Pro Asp Phe Thir Phe Glu Glin Wall Glin 195

Pro Glu Lell Lell Ala Ile Ser Gly Cys Lys Arg Asp Asp Asp 21 O 215 22O

Ser Ser Phe Pro Ala Phe Gly Asn Thir Thir Ser Asn Ala Lys 225 23 O 235 24 O

Pro Ser Gly Glu Lys Ser Ala Ala Ala Ala Glin Glin Pro Ser 245 250 255

Thir Ala Ala Thir Glu Pro Ile Ala Thir Ala Thir Wall Lys 26 O 265 27 O

Pro Ala Pro Wall Lys Pro Thir Lys Wall Wall ASn Pro Thir Ala 27s 285

Ser Lys Wall Gly Gly Thir Lys Thir Arg Gly Lys Cys Pro Ala Thir 29 O 295 3 OO

Pro Thir Pro Ala Ala Pro Glin Ser Ala Wall Ala Ile His 3. OS 310 315 32O

Glin Gly Gly Ser Ser Ala Pro Asp Gly Ser Luell Ser 3.25 330 335

Ala Ser Gly Ser Wall Met Asn Asp Ser Glin 34 O 345 35. O

Wall Pro Asn 355

<210s, SEQ ID NO 3 O &211s LENGTH: 376 212. TYPE : PRT <213> ORGANISM: Fusarium oxysporum

<4 OOs, SEQUENCE: 3 O

Met Arg Ser Tyr Thr Lell Lell Ala Luell Ala Gly Pro Lell Ala Wall Ser 1. 5 1O 15

Ala Ala Ser Gly Ser Gly His Ser Thir Arg Trp Asp Cys 25 3O

Pro Ser Cys Ser Trp Ser Gly Lys Ala Ala Wall Asn Ala Pro Ala Luell 35 4 O 45

Thir Cys Asp Lys Asn Asp Asn Pro Ile Ser ASn Thir Asn Ala Wall Asn US 9.238,806 B2 83 84 - Continued

SO 55 6 O

Gly Glu Gly Gly Gly Ser Ala Ala Cys Thir Asn Tyr Ser Pro 65 70

Trp Ala Wall Asn Asp Glu Lell Ala Gly Phe Ala Ala Thir Lys Ile 85 90 95

Ser Gly Gly Ser Glu Ala Ser Trp Cys Cys Ala Tyr Ala Lieu. Thir 105 11 O

Phe Thir Thir Gly Pro Wall Gly Met Ile Wall Glin Ser Thir 115 12 O 125

Asn Thir Gly Gly Asp Lell Gly Asp Asn His Phe Asp Leul Met Met Pro 13 O 135 14 O

Gly Gly Gly Wall Gly Ile Phe Asp Gly Thir Ser Glu Phe Gly Lys 145 150 155 160

Ala Luell Gly Gly Ala Glin Tyr Gly Gly Ile Ser Ser Arg Ser Glu 1.65 17O 17s

Asp Ser Pro Glu Lell Lell Asp Gly Cys His Trp Arg Phe Asp 18O 185 19 O

Trp Phe Glu Asn Ala Asp Asn Pro Asp Phe Thir Phe Glu Glin Wall Glin 195 2O5

Pro Ala Lell Lell Asp Ile Ser Gly Cys Lys Arg Asp Asp Asp 21 O 215 22O

Ser Ser Phe Pro Ala Phe Gly Asp Thir Ser Ala Ser Llys Pro Glin 225 23 O 235 24 O

Pro Ser Ser Ser Ala Thir Thir Ser Ala Ala Ala Ala Ala Glin 245 250 255

Pro Glin Thir Asp Ser Ala Pro Wall Wall Glin Llys Ser Ser Thir 26 O 265 27 O

Pro Ala Ala Glin Pro Glu Pro Thir Pro Ala Asp Llys Pro Glin 285

Thir Asp Pro Wall Ala Thir Pro Ala Ala Thir Llys Pro Ala Glin 29 O 295 3 OO

Pro Wall Asn Pro Lys Thir Thir Glin Wall Arg Gly Thr Lys Thir 3. OS 310 315

Arg Gly Ser Pro Ala Thir Asp Ala Thir Ala Lys Ala Ser Wall 3.25 330 335

Wall Pro Ala Tyr Tyr Glin Gly Gly Ser Ser Ala Tyr Pro Asn 34 O 345 35. O

Gly Asn Luell Ala Ala Thir Gly Ser Wall Lys Glin Asn Glu 355 360 365

Tyr Ser Glin Wall Pro Asn 37 O 375

SEQ ID NO 31 LENGTH: 349 TYPE : PRT ORGANISM: Acremonium sp.

< 4 OOs SEQUENCE: 31

Met Ile Ser Ala Trp Ile Lieu. Lieu. Gly Lieu Val Gly Ala Wall Pro Ser 1. 5 15

Ser Val Met Ala Ala Ser Gly Lys Gly His Thir Thr Arg Tyr Trp Asp 25 3O

Cys Cys Llys Thr Ser Cys Ala Trp Glu Gly Lys Ala Ser Wal Ser Glu 35 4 O 45 US 9.238,806 B2 85 86 - Continued

Pro Wall Luell Thir Cys Asn Lys Glin Asp Asn Pro Ile Val Asp Ala Asn SO 55 6 O

Ala Arg Ser Gly Cys Asp Gly Gly Gly Ala Phe Ala Cys Thir Asn Asn 65 70 7s

Ser Pro Trp Ala Wall Ser Glu Asp Luell Ala Gly Phe Ala Ala Thir 85 90 95

Ala Luell Ser Gly Gly Thir Glu Gly Ser Trp Ala Cys Ala 105 11 O

Ile Thir Phe Thir Ser Gly Pro Wall Ala Gly Met Wall Wall Glin 115 12 O 125

Ser Thir Asn Thir Gly Gly Asp Luell Ser Asn ASn His Phe Asp Luell Met 13 O 135 14 O

Ile Pro Gly Gly Gly Lell Gly Ile Phe Asp Gly Ser Ala Glin Phe 145 150 155 160

Gly Glin Luell Luell Pro Gly Glu Arg Gly Gly Wall Ser Ser Arg Ser 1.65 17O

Glin Asp Gly Met Pro Glu Luell Luell Asp Gly Glin Trp Arg 18O 185 19 O

Phe Asp Trp Phe Lys Asn Ser Asp Asn Pro Asp Ile Glu Phe Glu Glin 195 2O5

Wall Glin Pro Lys Glu Lell Ile Ala Wall Ser Gly Wall Arg Asp 21 O 215 22O

Asp Asp Ser Ser Phe Pro Wall Phe Glin Gly Ser Gly Ser Gly Asp Wall 225 23 O 235 24 O

Asn Pro Pro Pro Lys Pro Thr Thr Thr Thr Thr Ser Ser Pro Lys 245 250 255

Thir Thir Ser Ala Pro Ser Thir Luell Ser Asn Pro Ser Ala Pro Glin Glin 26 O 265 27 O

Pro Gly Asn Thir Asp Arg Pro Ala Glu Thir Thir Thir Thir Luell Pro 27s 285

Ala Luell Pro Ala Thir Thir Ser Ser Pro Ala Wall Ser Wall Pro Ser Ser 29 O 295 3 OO

Ser Ala Arg Wall Pro Lell Trp Gly Glin Asp Ser Glu Ala Ser Trp 3. OS 310 315

Asp Ala Pro Lys Ala Gly Thir Wall Wall Asn 3.25 330 335

Asp Trp Ser Glin Glin Pro Lys Asn Ser Ala 34 O 345

SEQ ID NO 32 LENGTH: 295 TYPE : PRT ORGANISM: Acremonium sp.

< 4 OOs SEQUENCE: 32

Met Arg Ser Thr Ser Ile Lell Ile Gly Luell Wall Ala Gly Wall Ala Ala 1. 5 15

Glin Ser Ser Gly Ser Gly His Thir Thir Arg Trp Asp Cys Lys 2O 25

Pro Ser Cys Ala Trp Asp Glu Lys Ala Ala Wall Ser Arg Pro Wall Thir 35 4 O 45

Thir Cys Asp Arg Asn Asn Ser Pro Luell Ser Pro Gly Ala Wall Ser Gly SO 55 6 O

Cys Asp Pro Asn Gly Wall Ala Phe Thir Cys ASn Asp Asn Glin Pro Trp 65 70 7s 8O US 9.238,806 B2 87 88 - Continued

Ala Wall Asn Asn. Asn Wall Ala Gly Phe Ala Ala Thir Ala Phe Pro 85 90 95

Gly Gly Asn Glu Ala Ser Trp Cys Ala Ala Luell Glin Phe 1OO 105 11 O

Thir Ser Gly Pro Wall Ala Gly Lys Thir Met Wall Wall Glin Ser Thir Asn 115 12 O 125

Thir Gly Gly Asp Lieu. Ser Gly Thir His Phe Asp Ile Glin Met Pro Gly 13 O 135 14 O

Gly Gly Luell Gly Ile Phe Asp Gly Thir Pro Glin Phe Gly Phe Thir 145 150 155 160

Phe Pro Gly Asn Arg Gly Gly Thir Thir Ser Arg Ser Glin Cys Ala 1.65 17s

Glu Luell Pro Ser Wall Lell Arg Asp Gly Cys His Trp Arg Tyr Asp Trp 18O 185 19 O

Phe Asn Asp Ala Asp Asn Pro Asn Wall Asn Trp Arg Arg Wall Arg 195

Pro Ala Ala Lieu. Thir Asn Arg Ser Gly Cys Wall Arg Asn Asp Asp Asn 21 O 215

Ser Pro Wall Phe Glu Pro Gly Thir Gly Thir Pro Pro Thir Pro Thir 225 23 O 235 24 O

Thir Thir Thir Thir Ser Ser Pro Pro Glin Pro Thir Asn Gly Gly Gly Gly 245 250 255

Gly Thir Ser Pro His Trp Gly Glin Cys Gly Gly Glin Gly Trp Ser Gly 26 O 265 27 O

Pro Thir Ala Cys Ala Gly Gly Ser Thir Cys ASn Lell Ile Asn Pro Trp 28O 285

Ser Glin Cys Ile Pro Asn 29 O 295

<210s, SEQ ID NO 33 &211s LENGTH: 225 212. TYPE : PRT &213s ORGANISM: Chrysosporium lucknowense

<4 OOs, SEQUENCE: 33

Met His Lieu. Ser Ala Thir Thir Gly Phe Luell Ala Lell Pro Ala Luell Ala 1. 5 15

Lell Ala Glin Luell Ser Gly Ser Gly Glin Thir Thir Arg Trp Asp 2O 25

Pro Ser Cys Ala Trp Pro Gly Lys Gly Pro Ser Ser Pro Wall 35 4 O 45

Glin Ala Asp Llys Asn Asp Asn Pro Luell ASn Asp Gly Gly Ser Thir SO 55 6 O

Arg Ser Gly Ala Gly Gly Ser Ala Tyr Met Ser Ser Glin 65 70 7s 8O

Ser Pro Trp Ala Wall Ser Asp Glu Luell Ser Gly Trp Ala Ala Wall 85 90 95

Luell Ala Gly Ser Ser Glu Ser Glin Trp Ala Cys Glu 1OO 105 11 O

Lell Thir Phe Thir Ser Gly Pro Wall Ala Gly Met Ile Wall Glin 115 12 O 125

Ala Thir Asn Thr Gly Gly Asp Luell Gly Asp ASn His Phe Asp Luell Ala 13 O 135 14 O

Ile Pro Gly Gly Gly Wall Gly Ile Phe Asn Ala Thir Asp Glin Tyr US 9.238,806 B2 89 - Continued

145 150 155 160 Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly Gly Ile His Ser 1.65 17O 17s Lys Glu Glu. Cys Glu Ser Phe Pro Glu Ala Lieu Lys Pro Gly Cys Asn 18O 185 19 O Trp Arg Phe Asp Trp Phe Glin Asn Ala Asp ASn Pro Ser Val Thr Phe 195 2OO 2O5 Gln Glu Val Ala Cys Pro Ser Glu Lieu. Thir Ser Lys Ser Gly Cys Ser 21 O 215 22O Arg 225

<210s, SEQ ID NO 34 &211s LENGTH: 225 212. TYPE: PRT <213> ORGANISM: Thiellavia heterothallica

<4 OOs, SEQUENCE: 34 Met His Leu Ser Ala Thr Thr Gly Phe Leu Ala Leu Pro Val Leu Ala 1. 5 1O 15 Lieu. Asp Gln Lieu. Ser Gly Ile Gly Glin Thir Thr Arg Tyr Trp Asp Cys 2O 25 3O Cys Llys Pro Ser Cys Ala Trp Pro Gly Lys Gly Pro Ser Ser Pro Val 35 4 O 45 Glin Ala Cys Asp Lys Asn Asp Asn Pro Lieu. Asn Asp Gly Gly Ser Thr SO 55 6 O Arg Ser Gly Cys Asp Ala Gly Gly Ser Ala Tyr Met Cys Ser Ser Glin 65 70 7s 8O Ser Pro Trp Ala Val Ser Asp Glu Lieu. Ser Tyr Gly Trp Ala Ala Val 85 90 95 Llys Lieu Ala Gly Ser Ser Glu Ser Glin Trp Cys Cys Ala Cys Tyr Glu 1OO 105 11 O Lieu. Thir Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Ile Val Glin 115 12 O 125 Ala Thr Asn. Thr Gly Gly Asp Lieu. Gly Asp Asn His Phe Asp Lieu Ala 13 O 135 14 O Ile Pro Gly Gly Gly Val Gly Ile Phe Asn Ala Cys Thr Asp Glin Tyr 145 150 155 160 Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly Gly Ile His Ser 1.65 17O 17s Lys Glu Glu. Cys Glu Ser Phe Pro Glu Ala Lieu Lys Pro Gly Cys Asn 18O 185 19 O Trp Arg Phe Asp Trp Phe Glin Asn Ala Asp ASn Pro Ser Val Thr Phe 195 2OO 2O5 Gln Glu Val Ala Cys Pro Ser Glu Lieu. Thir Ser Lys Ser Gly Cys Ser 21 O 215 22O

Arg 225

<210s, SEQ ID NO 35 &211s LENGTH: 338 212. TYPE: PRT <213> ORGANISM: Mucor circinelloides

<4 OOs, SEQUENCE: 35 Met Llys Phe Thr Val Ala Ile Thir Ser Ile Ala Val Ala Lieu Ala Lieu. US 9.238,806 B2 91 92 - Continued

15

Ser Ser Ser Ala Glu Ala Ala Ser Cys Ser Ser Wall Gly Gln Cys 25 3O

Gly Gly Ile Gly Trp Ser Gly Pro Thir Cys Glu Gly Ser Thr 35 4 O

Wall Ala Glin Glu Gly Asn Tyr Ser Glin Luell Pro Gly SO 55 6 O

Ser His Ser Asn Asn Ala Gly Asn Ala Ser Ser Thir Thir Ser 65 70 8O

Thir Thir Ser Thir Thir Thir Ala Ala Thir Ala Thir Wall Th Thr 85 90 95

Thir Wall Thir Lys Thir Thir Thir Lys Thir Thir Thir Thir Ser Thr 105 11 O

Thir Ala Ala Ala Ser Thir Ser Thir Ser Ser Ser Ala Gly Llys Val 115 12 O 125

Ile Ser Gly Gly Lys Ser Gly Ser Gly Ser Thir Thir Arg Trp Asp 13 O 135 14 O

Cys Ala Ser Cys Ser Trp Pro Gly Lys Ala Ser Wall Thr Gly 145 150 155 160

Pro Wall Asp Thir Cys Ala Ser Asn Gly Ile Ser Lell Lell Asp Ala Asn 1.65 17O 17s

Ala Glin Ser Gly Cys Asn Gly Gly Asn Gly Phe Met Asn Asn. Asn 18O 185 19 O

Glin Pro Trp Ala Wall Asn Asp Glu Luell Ala Gly Phe Ala Ala Ala 195

Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Gly Tyr Glu 21 O 215 22O

Lell Thir Phe Thir Ser Gly Ala Ala Ser Gly Lys Met Wall Wall Glin 225 23 O 235 24 O

Wall Thir Asn Thir Gly Gly Asp Luell Gly Ser ASn His Phe Asp Lieu. Glin 245 250 255

Met Pro Gly Gly Gly Wall Gly Ile Phe Asn Gly Ala Ala Gln Trp 26 O 265 27 O

Gly Ala Pro Asn Asp Gly Trp Gly Ala Arg Gly Gly Wall Ser Ser 27s 285

Wall Ser Asp Ala Ser Lell Pro Ser Ala Luell Glin Ala Gly 29 O 295 3 OO

Trp Arg Phe Asn Trp Phe Asn Ser Asp ASn Pro Thir Met Thir Phe 3. OS 310 315 32O

Lys Glu Wall Thir Cys Pro Ala Glu Luell Thir Thir Arg Ser Gly Cys Glu 3.25 330 335 Arg Lys

SEQ ID NO 36 LENGTH: 22O TYPE : PRT ORGANISM: Reticulitermes speratus hindgut protist

< 4 OOs SEQUENCE: 36

Met Lieu. Wall Phe Wall Phe Ser Lieu Lleu Ala Ser Wall Lell Phe Gly Asp 1. 5 15

Ser Gly Lys Thr Thr Arg Tyr Trp Asp Cys Cys Lys Gly Ser Cys Gly 25 3O

Trp Glu Ala Lys Ala Asp Wal Ser Llys Pro Ile Asp Thir Cys Ala Lys US 9.238,806 B2 93 94 - Continued

35 4 O 45

Asp Gly Thir Thir Wall Ala Ser Asn Asp Thir Wall Ser Gly SO 55 6 O

Asp Gly Gly Asp Gly Tyr Met Asp Glin Thir Pro Trp Gly Wall 65 70

Asn Asp Ser Ala Lell Gly Phe Ala Ala Ala Ala Ile Ser Gly Gly 85 90 95

Glu Ala Ala Cys Asn Tyr Glu Luell Thir Phe Thir Ser Gly 105 11 O

Pro Wall Asn Gly Met Thir Wall Glin Wall Thir Asn Thir Gly Gly 115 12 O 125

Asp Luell Gly Ser Asn Glin Phe Asp Luell Ala Ile Pro Gly Gly Gly Wall 13 O 135 14 O

Gly Ile Asn Gly Cys Thir Ala Glin Ser Gly Ala Pro Ala Asp Gly 145 150 155 160

Trp Gly Ser Arg Tyr Gly Gly Wall Ser Ser Arg Ser Glu Ser Glin 1.65 17O 17s

Lell Pro Ser Gly Lell Glin Ala Gly Cys Glin Trp Arg Phe Asp Trp Phe 18O 185 19 O

Glin Asn Ala Asp Asn Pro Ser Met Asn Phe ASn Wall Wall Ser Pro 195 2O5

Ser Glu Luell Ile Ala Thir Asn Arg Asn 21 O 215 22O

SEQ ID NO 37 LENGTH: 224 TYPE : PRT ORGANISM: Bursaphelenchus Xylophilus

< 4 OOs SEQUENCE: 37

Met Lys Ser Lieu Wall Phe Lell Ala Wall Luell Gly Lell Ala Wall Ala Glin 1. 5 15

Asp Thir Gly Lys Thir Thir Arg Trp Asp Pro Ser 25 3O

Ser Trp Pro Gly Ala Glin Luell Glin Gly Pro Ser Thir 35 4 O 45

Asp Wall Asn Asp Pro Lell Ser Asp Gly ASn Ile Glin Ser Gly SO 55 6 O

Asn Gly Gly Ser Ala Tyr Ala Ser Thir Asp Glin Pro Trp Ala 65 70

Asp Asp Asn Luell Ser Gly Phe Ala Ala Wall Lell Ala Gly 85 90 95

Glin Glu Ser Asp Trp Ser Cys Glu Lell Thir Phe Thir Asp 105 11 O

Gly Pro Wall Ala Gly Phe Wall Wall Glin Ala Thir Asn Thir Gly 115 12 O 125

Gly Asp Luell Gly Ser Asn His Phe Asp Luell Met Ile Pro Gly Gly Gly 13 O 135 14 O

Wall Gly Ile Phe Asn Gly Glin Ala Glin Trp Ser Pro Ala Glu 145 150 155 160

Gly Trp Gly Glin Arg Gly Gly Wall Ser Ser Ala Asp Cys Ala 1.65 17O 17s

Thir Luell Pro Thir Ala Lell Glin Pro Gly Cys ASn Trp Arg Phe Asp Trp 18O 185 19 O US 9.238,806 B2 95 - Continued Phe Lys Asn Ala Asp ASn Pro Gly Met Thr Phe Lys Arg Val Lys Cys 195 2OO 2O5 Pro Ala Glu Ile Thr Ala Lys Ser Gly Cys Ile Arg Ser Asp Asp Ala 21 O 215 22O

<210s, SEQ ID NO 38 &211s LENGTH: 355 212. TYPE: PRT <213> ORGANISM: Botryotinia fuckeliana

<4 OOs, SEQUENCE: 38 Met Glin Val Pro Met Lys Ser Leu Val Ala Leu Lleu Pro Phe Phe Leu 1. 5 1O 15 Glin Val Ser Ala Glin Ala Ser Gly Ser Gly. Thir Thr Thr Arg Tyr Trp 2O 25 3O Asp Cys Cys Llys Pro Ser Cys Ala Trp Ser Gly Lys Ala Thr Lieu. Glu 35 4 O 45 Ser Gly Ser Gly Pro Val Gly Thr Cys Asp Ile Asn Asp Ser Pro Leu SO 55 6 O Ser Asp Pro Thir Ala Ile Ala Val Ser Gly Cys Asp Gly Gly Asn. Ser 65 70 7s 8O Tyr Met Cys Ser Asp Glin Ser Pro Trp Ala Val Ser Asp Asp Lieu Ala 85 90 95 Tyr Gly Tyr Ala Ala Val Asn. Ile Ala Gly Gly Ser Glu Ala Ser Trp 1OO 105 11 O Cys Cys Ala Cys Tyr Glu Lieu. Thr Phe Thr Ser Thr Ala Leu Ala Gly 115 120 125 Llys Llys Met Ile Val Glin Ala Thr Asn Thr Gly Gly Asp Lieu. Gly Ser 13 O 135 14 O Asn Glin Phe Asp Lieu Ala Ile Pro Gly Gly Gly Val Gly Ile Phe Asn 145 150 155 160 Gly Cys Thr Lys Glu Phe Gly Ala Pro Ser Ser Gly Trp Gly Ala Glin 1.65 17O 17s Tyr Gly Gly Val Ala Ala Val Ser Ser Cys Ala Ala Phe Pro Glu Ala 18O 185 19 O Lieu Lys Pro Gly Cys Ser Phe Arg Phe Asp Trp Phe Glu Gly Ala Asp 195 2OO 2O5 Asn Pro Thr Val Asn Phe Lys Glin Val Asn Cys Pro Ala Glu Lieu. Thr 21 O 215 22O Llys Ser Thr Gly Cys Lys Arg Ala Asp Asp Ser Ser Met Pro Ala Pro 225 23 O 235 24 O Asp Ala Ser Gly Ser Ala Ser Ala Ser Pro Val Ala Ser Thir Ser Ala 245 250 255 Lys. Thir Ser Ser Val Ala Pro Thr Ser Val Ser Ser Ser Ser Val Val 26 O 265 27 O

Wall Ala Pro Ser Ser Ala Thir Ser Ser Pro Wal Wal Wal Wall Pro Thr 27s 28O 285

Ser Ala Ala Ser Ser Lys Ala Ser Ser Ala Ala Val Val Ser His Pro 29 O 295 3 OO

Wal Wall Pro Ser Ser Glu Ala Ser Ser Ala Pro Ala Wall Thir Ser His 3. OS 310 315 32O

Ser Ser Ala Thr Lys Ser Ala Lys Thr Ser Val Ala Ala Pro His Ser 3.25 330 335

Thir Ser Ala Ser Thr Gly Tyr Gly Ser Gly Asp Asp Asp Asp Thr Cys 34 O 345 35. O US 9.238,806 B2 97 98 - Continued

Asp Ala Glu 355

<210s, SEQ ID NO 39 &211s LENGTH: 251 212. TYPE : PRT &213s ORGANISM: Acremonium thermophilum

<4 OOs, SEQUENCE: 39

Met Arg Lieu Pro Lell Pro Thir Luell Luell Ala Luell Lell Pro Tyr Luell 1. 5 15

Glu Wall Ser Ala Glin Gly Ala Ser Gly Thir Gly Thir Thir Thir Arg 2O 25

Trp Asp Cys Lys Pro Ser Cys Ala Trp Pro Lell Lys Gly Asn Ser 35 4 O 45

Pro Ser Pro Wall Glin Thir Cys Asp Asn Asp Arg Pro Luell Asn Asp SO 55 6 O

Gly Gly Asn Thir Ser Gly Asp Asn Gly Gly Gly Ala Phe Met 65 70

Ser Ser Glin Ser Pro Trp Ala Wall Asn Glu Thir Thir Ser Tyr Gly 85 90 95

Trp Ala Ala Wall Arg Ile Ala Gly Ser Thir Glu Ser Ala Trp 105 11 O

Ala Tyr Glu Lell Thir Phe Thir Ser Gly Pro Wall Ser Gly 115 12 O 125

Lieu Ile Wall Gln Ala Thr Asn Thr Gly Gly Asp Lieu Gly Ser Asn His 13 O 135 14 O

Phe Asp Luell Ala Ile Pro Gly Gly Gly Wall Gly Glin Ser Asn Ala Cys 145 150 155 160

Thir Asn Glin Tyr Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly 1.65 17O 17s

Gly Wall His Ser Arg Ser Asp Asp Ser Phe Pro Ala Ala Luell 18O 185 19 O

Ala Gly Cys Trp Arg Phe Asp Trp Phe Glin Gly Ala Asp Asn Pro 195 2OO

Ser Wall Ser Phe Glin Wall Ala Pro Ala Ala Ile Thir Ala 21 O 215

Ser Gly Thir Glin Asn Asp Ala Ile ASn Glu Thir Pro Thir Gly 225 23 O 235 24 O

Pro Ser Thir Wall Pro Thir Thir Ala Ser Gly 245 250

<210s, SEQ ID NO 4 O &211s LENGTH: 229 212. TYPE : PRT &213s ORGANISM: Scopulariopsis brevicaulis

<4 OOs, SEQUENCE: 4 O

Met His Wall Lieu. Pro Thir Lell Luell Ala Lieu. Thir Pro Lell Wall Luell Pro 1. 5 15

Ala Ala Ser Glin Ala Ser Gly Thir Gly. Thir Thr Thir Arg Tyr Trp Asp 25 3O

Cys Cys Llys Pro Ser Ser Trp Pro Asp Llys Ala Pro Luell Ser Glin 35 4 O 45

Gly Pro Pro Met Thir Asp Ile Asn Asp Asn Pro Lell Asp Asp Gly SO 55 6 O US 9.238,806 B2 99 100 - Continued

Gly Luell Thir Glu Ser Gly Glu Pro Gly Gly Gly Ala Met Cys 65 70

Ser Ser His Ser Pro Trp Ala Wall Asp Asp Glu Lell Ala Gly Trp 85 90 95

Ala Ala Wall Asn Ile Gly Gly Glin Thir Glu Ser Asp Trp Cys Ala 105 11 O

Glu Luell Glu Phe Thir Thir Gly Ala Wall Ser Gly Met 115 12 O 125

Ile Wall Glin Ala Thir Asn Thir Gly Gly Asp Luell Gly Asn Asn His Phe 13 O 135 14 O

Asp Ile Ala Met Pro Gly Gly Gly Wall Gly Ile Phe Asn Gly Thir 145 150 155 160

Asp Glin Trp Gly Ser Pro Pro Asn Gly Trp Gly Asp Arg Gly Gly 1.65 17O 17s

Wall His Thir Arg Ala Asp Asp Ser Phe Pro Glu Ala Luell Ala 18O 185 19 O

Gly Glu Trp Arg Phe Asp Trp Phe Gly Gly Thir Asp Asn Pro Asp 195 2OO

Wall Ser Phe Arg Glu Wall Glu Pro Ala Glu Lell Wall Glin Ser 21 O 215 22O

Glin Glin Arg Ser 225

SEQ ID NO 41 LENGTH: 34 O TYPE : PRT ORGANISM: Syncephalastrum ace Sl

< 4 OOs SEQUENCE: 41

Met Ile Lieu. His Arg Thir Glu Asn Asn Asp Met Arg Luell Ser Phe 1. 5 15

Ala Ala Ser Luell Lell Lell Ala Thir Wall Gly Met Glin Lell Wall Ser Ala 25

Ala Asp Cys Ser Asn Ala Tyr Ser Glin Cys Gly Gly Glin Asn Trp 35 4 O 45

Ser Gly Glu Ser Wall Ser Gly Tyr Glu Cys Glin Luell Asn SO 55 6 O

Asp His Cys Wall Pro Glin Asn Ser Gly Ser Phe Ser Gly 65 70

Ser Ser Ser Ala Ala Pro Ser His Met Ala Thir Ser Ser Ala Pro 90 95

Ser Ser Ser Lys Pro Ser Ser Pro Ala Ser Ser Ser Lys Thir Pro 105 11 O

Ser Ser Pro Ala Ser Ser Ser Ser Ser Ser Gly Ser Gly Tyr 115 12 O 125

Pro Ile Ser Gly Ala Ser Gly Asp Gly Thir Thir Thir Arg Trp 13 O 135 14 O

Asp Ser Ser Trp Pro Gly Lys Ala Pro Wall Thir 145 150 155 160

Asn Pro Wall Gly Thir Ala Asp Gly Wall Lell Wall Asp Ala 1.65 17O 17s

Asn Wall Glin Ser Gly Asn Gly Gly Glu Gly Met Cys Asn Asp 18O 185 19 O

Asn Glin Pro Trp Ala Ile Asp Asp Asn Luell Ser Gly Phe Ala Ala US 9.238,806 B2 101 102 - Continued

195 2OO 2O5

Ala Ser Ile Ser Gly Ser Glu Ser Asp Phe Cys Ser 21 O 215 22O

Glu Luell Thir Phe Ser Ser Gly Glu Ile Glu Gly Lys Met Wall Wall 225 23 O 235 24 O

Glin Wall Thir Asn Thir Gly Gly Asp Luell Ser ASn Asn His Phe Asp Luell 245 250 255

Glin Ile Pro Gly Gly Gly Wall Gly Ile Phe ASn Gly Glin Thir Glin 26 O 265 27 O

Trp Asp Ala Pro Ser Asp Gly Trp Gly Glin Arg Gly Gly Ile Ser 285

Ser Ala Ser Glu Ser Glin Luell Pro Glin Lell Glin Asp Gly 29 O 295 3 OO

Lys Trp Arg Phe Asp Trp Phe Asn Ala Asp Asn Pro Asn Wall Ser 3. OS 310 315 32O

Phe Glin Wall Ser Pro Ala Glu Luell Wall Thir Gly 3.25 330 335

Glu Arg Thir Ser 34 O

<210s, SEQ ID NO 42 &211s LENGTH: 338 212. TYPE : PRT &213s ORGANISM: Rhizopus oryzae

<4 OOs, SEQUENCE: 42

Met Llys Phe Ile Thir Ile Ala Ser Ser Ala Luell Lell Ala Luell Ala Luell 1. 5 15

Gly Thir Glu Met Ala Ser Ala Ala Glu Ser Lell Tyr Gly Glin 25

Cys Gly Gly Asn Trp Asn Gly Pro Thir Glu Ser Gly Ser 35 4 O 45

Thir Cys Wall Ser Asn Asp Ser Glin Lell Pro Ser Gly SO 55

Ser Ser Gly Asn Ser Ser Glu Ser Ala His Thir Thir Thir 65 70 7s

Ala Ala His Lys Thir Thir Thir Ala Ala His Thir Thir Thir 85 90 95

Ala Pro Ala Lys Lys Thir Thir Thir Wall Ala Ala Ser Thir Pro Ser 1OO 105 11 O

Asn Ser Ser Ser Ser Ser Ser Gly Lys Tyr Ser Ala Wall Ser Gly Gly 115 12 O 125

Ala Ser Gly Asn Gly Wall Thir Thir Arg Tyr Trp Asp Ala 13 O 135 14 O

Ser Ser Trp Pro Gly Lys Ala Asn Wall Ser Ser Pro Wall Ser 145 150 155 160

Asn Asp Gly Wall Thir Ala Luell Ser Asp Ser Asn Ala Glin Ser 1.65 17O 17s

Gly Asn Gly Gly Asn Ser Met Cys ASn Asp Asn Glin Pro Trp 18O 185 19 O

Ala Wall Asn Asp Asn Lell Ala Tyr Gly Phe Ala Ala Ala Ala Ile Ser 195 2OO

Gly Gly Gly Glu Ser Arg Trp Ser Cys Phe Glu Luell Thir Phe 21 O 215 22O US 9.238,806 B2 103 104 - Continued

Thir Ser Thir Ser Val Ala Gly Llys Llys Met Wall Wall Glin Wall. Thir Asn 225 23 O 235 24 O

Thir Gly Gly Asp Lieu. Gly Ser Ser Thr Gly Ala His Phe Asp Luell Glin 245 250 255

Met Pro Gly Gly Gly Val Gly Ile Phe Asn Gly Ser Ser Glin Trp 26 O 265 27 O

Gly Ala Pro Asn Asp Gly Trp Gly Ser Arg Gly Gly Ile Ser Ser 27s 28O 285

Ala Ser Asp Ser Ser Leu. Pro Ser Ala Luell Glin Ala Gly 29 O 295 3 OO

Trp Arg Phe Asn Trp Phe Lys Asn Ala Asp ASn Pro Ser Met Thir Tyr 3. OS 310 315 32O

Lys Glu Wall Thir Cys Pro Llys Glu Ile Thr Ala Thir Gly Cys Ser 3.25 330 335 Arg Lys

<210s, SEQ ID NO 43 &211s LENGTH: 226 212. TYPE : PRT &213s ORGANISM: Crinipellis scabella

<4 OOs, SEQUENCE: 43

Met Wal His Pro Asn Met Luell Llys Thr Lieu. Ala Pro Lell Ile Ile Luell 1. 5 1O 15

Ala Ala Ser Wall Thir Ala Glin Thr Ala Gly Wall Thir Thir Arg Trp 25 3O

Asp Cys Pro Ser Cys Gly Trp Ser Gly Ala Ser Wall Ser 35 4 O 45

Ala Wall Arg Thr Cys Asp Arg Asn Gly ASn Thir Lell Gly Pro Asp 55 6 O

Wall Ser Gly Cys Asp Ser Gly Gly Thr Ser Phe Thir Ala Asn 65 70

Asn Gly Pro Phe Ala Ile Asp Asn Asn. Thr Ala Gly Phe Ala Ala 85 90 95

Ala His Luell Ala Gly Ser Ser Glu Ala Ala Trp Glin Tyr 105 11 O

Glu Luell Thir Phe Thir Ser Gly Pro Wal Wall Gly Lys Luell Thir Wall 115 12 O 125

Glin Wall Thir Asn Thr Gly Gly Asp Lieu. Gly ASn Asn His Phe Asp Luell 13 O 135 14 O

Met Ile Pro Gly Gly Gly Val Gly Leu Phe Thir Glin Gly Pro Ala 145 150 155 160

Glin Phe Gly Ser Trp Asn Gly Gly Ala Glin Tyr Gly Gly Wall Ser Ser 1.65 17O 17s

Arg Asp Glin Cys Ser Glin Lieu. Pro Ala Ala Wall Glin Ala Gly Glin 18O 185 19 O

Phe Arg Phe Asp Trp Met Gly Gly Ala Asp ASn Pro Asn Wall Thir Phe 195 2OO 2O5

Arg Pro Wall Thir Cys Pro Ala Gln Lieu. Thir ASn Ile Ser Gly Wall 21 O 215 22O

Arg 225

<210s, SEQ ID NO 44 &211s LENGTH: 222 US 9.238,806 B2 105 106 - Continued

212. TYPE: PRT <213> ORGANISM: Macrophomina phaseolina

<4 OOs, SEQUENCE: 44 Met Phe Ser Pro Lieu. Trp Ala Lieu. Ser Ala Lieu Lleu Lieu. Phe Pro Ala 1. 5 1O 15 Thr Glu Ala Thr Ser Gly Val Thir Thr Arg Tyr Trp Asp Cys Cys Lys 2O 25 3O Pro Ser Cys Ala Trp Thr Gly Lys Ala Ser Val Ser Lys Pro Val Gly 35 4 O 45 Thir Cys Asp Ile Asn Asp Asn Ala Glin Thr Pro Ser Asp Lieu. Lieu Lys SO 55 6 O Ser Ser Cys Asp Gly Gly Ser Ala Tyr Tyr Cys Ser Asn Glin Gly Pro 65 70 7s 8O Trp Ala Val Asn Asp Ser Lieu. Ser Tyr Gly Phe Ala Ala Ala Lys Lieu. 85 90 95 Ser Gly Lys Glin Glu Thir Asp Trp Cys Cys Gly Cys Tyr Lys Lieu. Thr 1OO 105 11 O Phe Thir Ser Thr Ala Val Ser Gly Lys Glin Met Ile Val Glin Ile Thr 115 12 O 125 Asn Thr Gly Gly Asp Lieu. Gly Asn. Asn His Phe Asp Ile Ala Met Pro 13 O 135 14 O Gly Gly Gly Val Gly Ile Phe Asin Gly Cys Ser Lys Gln Trp Asn Gly 145 150 155 160 Ile Asn Lieu. Gly Asn. Glin Tyr Gly Gly Phe Thr Asp Arg Ser Glin Cys 1.65 170 175 Ala Thr Lieu Pro Ser Llys Trp Glin Ala Ser Cys Asn Trp Arg Phe Asp 18O 185 19 O Trp Phe Glu Asn Ala Asp Asn Pro Thr Val Asp Trp Glu Pro Val Thr 195 2OO 2O5 Cys Pro Glin Glu Lieu Val Ala Arg Thr Gly Cys Ser Arg Thr 21 O 215 22O

<210s, SEQ ID NO 45 &211s LENGTH: 245 212. TYPE: PRT <213> ORGANISM: Podospora anserina

<4 OOs, SEQUENCE: 45 Met Glin Lieu Ala Lieu. Thir Ile Lieu Ala Phe Gly Gly Phe Ala Ser Ala 1. 5 1O 15 Glin Gly Ala Glin Gly Ala Gly Lys Thir Thr Arg Tyr Trp Asp Cys Cys 2O 25 3O Llys Pro Ser Cys Ala Trp Pro Gly Lys Ser Thr Ala Ser Thr Pro Val 35 4 O 45 Lieu. Thir Cys Asp Arg Asn Asp Asin Pro Lieu. Asn Asp Arg Gly Ser Thr SO 55 6 O

Arg Ser Gly Cys Asp Ser Gly Gly Ser Ala Phe Met Cys Ser Asn Glin 65 70 7s 8O

Ser Pro Trp Ala Val Asn Glu Thr Val Ala Tyr Gly Trp Ala Ala Val 85 90 95

Asn. Ile Ala Gly Ser Asn. Glu Ala Ser Trp Cys Cys Ser Cys Tyr Glu 1OO 105 11 O

Lieu. Thir Phe Thr Ser Gly Pro Val Ser Gly Lys Lys Met Ile Val Glin 115 12 O 125 US 9.238,806 B2 107 108 - Continued

Ala Thir Asn Thir Gly Gly Asp Luell Gly Asn ASn His Phe Asp Ile Ala 13 O 135 14 O

Met Pro Gly Gly Gly Wall Gly Ile Phe Asn Ala Cys Thir Glin Glin Tyr 145 150 155 160

Gly Ala Pro Pro Asn Gly Trp Gly Glu Arg Tyr Gly Gly Wall Gly Ser 1.65 17s

Ser Ala Cys Glu Ser Phe Pro Asp Luell Ala Gly Cys Asn 185 19 O

Trp Arg Phe Asp Trp Phe Met Gly Ala Asp ASn Pro Asp Wall Arg Phe 195

Arg Glin Wall Ala Pro Ala Ala Ile Thir Ala Lys Ser Glin Wall 21 O 215 22O

Arg Glin Arg Asp Wall Ile Asp Glin Thir Pro Thir Gly Pro Ser Thir Wall 225 23 O 235 24 O

Pro Thir Trp Thir Pro 245

SEQ ID NO 46 LENGTH: 366 TYPE : PRT ORGANISM: Rhizopus oryzae

< 4 OOs SEQUENCE: 46

Met Llys Phe Ile Thir Ile Thir Ser Ser Ala Luell Lell Ala Luell Ala Luell 1. 5 15

Gly Thir Glu Met Ala Ser Ala Ala Lys Ser Lell Tyr Gly Glin 25 30

Cys Gly Gly Asp Trp Asn Gly Pro Thir Cys Glu Ser Gly Ser 35 4 O 45

Thir Cys Wall Ser Asn Asp Ser Glin Cys Lell Ala Pro Glu SO 55 6 O

Ser Asn Asn Ser Ser Glu Ser Lys Lell Tyr Gly Glin Cys 65 70 7s

Gly Gly Asp Trp Asn Gly Pro Thir Cys Glu Ser Gly Ser Thir 85 90 95

Wall Ser Asn Asp Ser Glin Lell Ala Pro Glu Ser 1OO 105 11 O

Asn Gly Asn Thir Ser Glu Ser Ala His Thir Thir Thir Thir Thir 115 12 O 125

Ala Pro Ala Glu Ile Thir Thir Thir Ala Lys Ala Ser Asn Ser Ser 13 O 135 14 O

Asn Ser Ser Gly Tyr Ser Ile Wall Ser Gly Gly Ala Ser Gly Asn 145 150 155 160

Gly Wall Thir Thir Arg Trp Asp Cys Ala Ser Ser Trp 1.65 17s

Pro Gly Ala Asn Wall Ser Ser Pro Wall Lys Ser Asn Asp 18O 185 19 O

Gly Wall Thir Ala Lell Ser Asp Ser Asn Wall Glin Ser Gly Cys Asn Gly 195 2OO 2O5

Gly Asn Ser Met Asn Asp Asn Glin Pro Trp Ala Wall Asn Asp 21 O 215

Asn Luell Ala Gly Phe Ala Ala Ala Ala Ile Ser Gly Gly Gly Glu 225 23 O 235 24 O

Ser Arg Trp Cys Ser Phe Glu Luell Thir Phe Thir Ser Thir Ser 245 250 255 US 9.238,806 B2 109 110 - Continued

Wall Ala Gly Lys Lys Met Wall Ile Glin Wall Thir Asn Thir Gly Gly Asp 26 O 265 27 O

Lell Gly Ser Ser Thir Gly Ala His Phe Asp Luell Glin Met Pro Gly Gly 27s 28O 285

Gly Wall Gly Ile Phe Asn Gly Ser Lys Glin Trp Gly Ala Pro Asn 29 O 295 3 OO

Asp Gly Trp Gly Ser Arg Gly Gly Ile Ser Ser Ala Ser Asp Cys 3. OS 310 315

Ser Ser Luell Pro Ser Ala Lell Glin Ala Gly Cys Trp Arg Phe Asn 3.25 330 335

Trp Phe Asn Ala Asp Asn Pro Ser Met Thir Glu Wall Thir 34 O 345 35. O

Pro Lys Glu Ile Thir Ala Lys Thir Gly Cys Ser Arg 355 360 365

SEO ID NO 47 LENGTH: 286 TYPE : PRT ORGANISM: Bursaphelenchus Xylophilus

< 4 OOs SEQUENCE: 47

Met Ala Lys Lieu Phe Wall Ser Wall Luell Wall Wall Ala Lell Luell Luell Glu 1. 5 15

Wall Wall Wall Glu Cys Ser Ser Lys Thir Thir Lys Ala Thir 2O 25

Thr Wall Lys Ser Thr Ala Thr Ala Ala Pro Thr Lys Ala Thr Ala 35 4 O 45

Ala Pro Thir Ala Thir Thir Ala Ala Ser Ser Ser Ala Gly Gly Asn SO 55 6 O

Ser Ala Asp Ser Tyr Ser Wall Ser Gly Gly Thir Ser Gly Thir 65 70

Gly Wall Thir Thir Trp Asp Cys Ser Ser Ala Trp 85 90 95

Pro Gly Ala Ser Lell Ser Gly Pro Ile Glin Thir Cys Asp Wall 105 11 O

His Asp Glin Pro Lell Asn Asp Gly Gly Asn Thir Glin Ser Gly Asn 115 12 O 125

Gly Gly Ser Ala Tyr Ser Cys Ser Thir Glu Glin Pro Ala Wall Asn 13 O 135 14 O

Asp Thir Luell Ser Phe Gly Phe Ala Ala Wall Lys Lell Ala Gly Gly Ser 145 150 155 160

Glu Ser Ser Trp Cys Ala Glu Luell Thir Phe Thir Ser Gly 1.65 17O 17s

Ser Wall Asn Gly Lys Phe Wall Ile Glin Ala Thir Asn Thir Gly Gly 18O 185 19 O

Asp Luell Gly Asp Asn His Phe Asp Luell Ala Ile Pro Gly Gly Gly Wall 195

Gly Ile Phe Asn Gly Thir Ala Glin Trp Gly Ala Pro Ser Ser Gly 21 O 215

Trp Gly Ala Glin Tyr Gly Gly Wall Ser Ser Arg Ser Asp Ser Glin 225 23 O 235 24 O

Lell Pro Ala Lell Glin Pro Gly Asp Trp Arg Phe Asp Trp Phe 245 250 255

Gly Asn Ser Asp Asn Pro Gly Wall Thir Phe Glin Wall Thir Pro US 9.238,806 B2 111 112 - Continued

26 O 265 27 O Llys Thr Lieu. Thir Asp Llys Ser Lys Cys Ile Arg Ala Asp Asp 285

<210s, SEQ ID NO 48 &211s LENGTH: 359 212. TYPE : PRT <213> ORGANISM: Bursaphelenchus Xylophilus

<4 OOs, SEQUENCE: 48

Met Asn Llys Lieu Lell Wall Ser Wall Luell Wall Luell Ala Lell Luell Phe Glu 1. 5 15

Asn Wall Wall Glu Gly Thir Gly Pro Luell Thir Thir Ala Ala Gly 25

Ala Thir Glin Thir Ala Pro Pro Ala Ser Ala Ala Ser Thir Thir Gly 35 4 O 45

Glin Thir Ala Ala Ser Gly Ser Pro Ala Thir Thir Ala Ser Ala Ala Pro SO 55 6 O

Thir Ala Ser Ala Ala Pro Ser Thir Ala Ser Ala Ala Pro Ser Lys 65 70

Ala Ser Ala Ala Pro Ser Thir Ala Ser Ala Ala Pro Ser Thir Ala Ser 85 90 95

Ala Ala Pro Ser Ala Ser Ala Ala Pro Ser Thir Ala Ser Ser Thir 105 11 O

Ala Ser Ser Thir Thir Pro Thir Thir Thir Thir Ala Ser Ser Thir Ala 115 12 O 125

Ala Ala Thir Thir Glin Ala Ser Gly Asn Ser Ala Asp Ser Ser 13 O 135 14 O

Tyr Ile Ser Gly Gly Thir Ser Gly Thir Gly Wall Thir Thir Arg Trp 145 150 155 160

Asp Ser Ser Ala Trp Pro Gly Ala Thir Luell 1.65 17O 17s

Ser Gly Pro Ile Glin Thir Asp Wall His Asp Glin Pro Luell Asn 18O 185 19 O

Gly Gly Asn Thir Glin Ser Gly Cys Asn Gly Gly Ser Ala Ser 195

Ser Thir Glu Glin Pro Ala Wall Asn Asp Thir Lell Ser Phe Gly Phe 21 O 215

Ala Ala Wall Lell Ala Gly Gly Ser Glu Ser Thir Trp Ala 225 23 O 235 24 O

Glu Luell Thir Phe Thir Ser Gly Ser Wall Ala Gly Lys Phe 245 250 255

Wall Ile Glin Ala Thir Asn Thir Gly Gly Asp Luell Gly Asp Asn His Phe 26 O 265 27 O

Asp Luell Ala Ile Pro Gly Gly Gly Wall Gly Ile Phe Asn Gly Thir 28O 285

Ala Glin Trp Gly Ala Pro Ser Ser Gly Trp Gly Ser Glin Tyr Gly Gly 29 O 295 3 OO

Wall Ser Ser Arg Ser Asp Ser Glin Luell Pro Ala Thir Luell Glin Pro 3. OS 310 315

Gly Asp Trp Arg Phe Asp Trp Phe Gly ASn Ser Asp Asn Pro Gly 3.25 330 335

Wall Thir Phe Lys Glin Wall Thir Pro Thir Ile Thir Asp Ser 34 O 345 35. O US 9.238,806 B2 113 114 - Continued Lys Cys Ile Arg Ala Asp Asp 355

<210s, SEQ ID NO 49 &211s LENGTH: 227 212. TYPE: PRT <213> ORGANISM: Humicola grisea var. thermoidea <4 OOs, SEQUENCE: 49

Met Glin Lieu. Pro Lieu. Thir Thir Lieu. Lieu. Thir Lieu. Lieu Pro Ala Lieu Ala 1. 5 1O 15 Ala Ala Glin Ser Gly Ser Gly Arg Thir Thr Arg Tyr Trp Asp Cys Cys 2O 25 3O Llys Pro Ser Cys Ala Trp Pro Gly Lys Gly Pro Ala Pro Val Arg Thr 35 4 O 45 Cys Asp Arg Trp Asp ASn Pro Lieu. Phe Asp Gly Gly Asn. Thir Arg Ser SO 55 6 O Gly Cys Asp Ala Gly Gly Gly Ala Tyr Met Cys Ser Asp Glin Ser Pro 65 70 7s 8O Trp Ala Val Ser Asp Asp Lieu Ala Tyr Gly Trp Ala Ala Val Asn. Ile 85 90 95 Ala Gly Ser Asn. Glu Arg Glin Trp Cys Cys Ala Cys Tyr Glu Lieu. Thr 1OO 105 11 O Phe Thir Ser Gly Pro Val Ala Gly Lys Arg Met Ile Val Glin Ala Ser 115 12 O 125 Asn Thr Gly Gly Asp Lieu. Gly Asn. Asn His Phe Asp Ile Ala Met Pro 13 O 135 14 O Gly Gly Gly Val Gly Ile Phe Asn Ala Cys Thr Asp Glin Tyr Gly Ala 145 150 155 160 Pro Pro Asn Gly Trp Gly Glin Arg Tyr Gly Gly Ile Ser Glin Arg His 1.65 17O 17s Glu Cys Asp Ala Phe Pro Glu Lys Lieu Lys Pro Gly Cys Tyr Trp Arg 18O 185 19 O Phe Asp Trp Cys Val Ser Leu Phe Pro Pro Leu Ser Leu Ser Leu Pro 195 2OO 2O5 Pro Gly Thr Gly Glin Thr Met Gly Arg Ser Cys Val Phe Phe Pro Leu 21 O 215 22O

Ser Ala Asn 225

<210s, SEQ ID NO 50 &211s LENGTH: 346 212. TYPE: PRT <213> ORGANISM: Phycomyces initens

<4 OOs, SEQUENCE: 50 Met Llys Phe Ser Ile Ile Ala Ser Ala Lieu. Lieu. Lieu Ala Ala Ser Ser 1. 5 1O 15 Thr Tyr Ala Ala Glu. Cys Ser Glin Gly Tyr Gly Glin Cys Gly Gly Lys 2O 25 3O

Met Trp Thr Gly Pro Thr Cys Cys Thr Ser Gly Phe Thr Cys Val Gly 35 4 O 45

Ala Glu Asn. Asn. Glu Trp Tyr Ser Glin Cys Ile Pro Asn Asp Glin Val SO 55 6 O

Gln Gly Asn Pro Llys Thr Thr Thr Thr Thr Thr Thr Lys Ala Ala Thr 65 70 7s 8O US 9.238,806 B2 115 116 - Continued

Thir Thir Ala Pro Wall Thir Thir Lys Ala Thir Thir Thir Thir Thir 85 90 95

Thir Ala Pro Wall Thir Thir Lys Ala Thir Thir Thir Thir Thir Thir 105 11 O

Thir Thir Thir Thir Thir Thir Ala Ala Thir Thir Thir Ser 115 12 O 125

Ser Ser Asn Thir Tyr Pro Ile Ser Gly Gly Phe Ser Gly Asn 13 O 35 14 O

Gly Arg Thir Thir Tyr Asp Lys Pro Ser Ala Trp 145 150 155 160

Asp Gly Ala Ser Wall Pro Wall Luell Thir Ala Lys Asp 1.65 17O 17s

Gly Wall Ser Arg Lell Gly Ser Asp Wall Glin Ser Gly Wall Gly Gly 18O 185 19 O

Glin Ala Tyr Met Cys Asn Asp Asn Glin Pro Trp Wall Wall Asn Asp Asp 195

Lell Ala Gly Phe Ala Ala Ala Ser Luell Gly Ser Ala Gly Ala Ser 21 O 215 22O

Ala Phe Gly Cys Glu Luell Thir Phe Thir Asn Thir Ala Wall 225 23 O 235 24 O

Ala Gly Phe Wall Wall Glin Wall Thir ASn Thir Gly Asp Asp Luell 245 250 255

Ser Thir Asn His Phe Asp Lell Glin Met Pro Gly Gly Gly Wall Gly 26 O 265 27 O

Phe Asn Gly Gln Ser Gln Trp Asn Thr Asn Thr Asp Gly Trp Gly 27s 285

Ala Arg Gly Gly Ile Ser Ser Ile Ser Glu Cys Asp Luell Pro 29 O 295 3 OO

Thir Glin Luell Glin Ala Gly Trp Arg Phe Gly Trp Phe Asn 3. OS 310 315

Ala Asp Asn Pro Glu Wall Thir Phe Ala Wall Thir Pro Ala Glu 3.25 330 335

Ile Ile Ala Lys Thir Gly Glu Arg 34 O 345

SEO ID NO 51 LENGTH: 360 TYPE : PRT ORGANISM: Rhizopus oryzae

< 4 OOs SEQUENCE: 51

Met Llys Phe Leu Thir Ile Ala Ser Ser Ala Ile Lell Ala Luell Ala Wall 1. 5 1O 15

Gly Thir Glu Met Ala His Ala Ala Glu Ser Ala Tyr Glin 2O 25 3O

Cys Gly Gly Asn Trp Asp Gly Pro Thir Glu Ser Gly Ser 35 4 O 45

Thir Cys Wall Asp Tyr Pro Asp Asn Pro Phe Ser Glin Wall Pro SO 55 6 O

Asn Glu Asn Luell Thir Ser Thir Asn Ser His Thir Thir Thir 65 70

Thir Glu Ser Ala Lys Thir Thir Thir Thir Gly Ser Lys Thir 85 90 95

Thir Thir Thir Glu Ala Ser Thir Thir Thir Thir Glu Ala Ser 1OO 105 11 O US 9.238,806 B2 117 118 - Continued

Thir Thir Thir Thir Glu Ala Ser Thir Thir Thir Thir Thir 115 12 O 125

Ala Ser Thir Ser Thir Ser Ser Ser Ser Ser Ser Ala Ser Thir Asn 13 O 135 14 O

Tyr Ser Ala Wall Ser Gly Gly Ala Ser Gly ASn Gly Glu Thir Thir Arg 145 150 155 160

Trp Asp Cys Pro Ser Ser Trp Pro Gly Ala Asp 1.65 17O

Wall Thir Ser Pro Wall Gly Ser Asn Asp Gly Thir Luell Ala 18O 185 19 O

Asp Asn Asn Thir Glin Asn Gly Cys Wall Gly Gly Ser Ser Thir 195

Asn Asp Asn Glin Pro Trp Wall Wall Ser Asp Asp Lell Ala Gly Phe 21 O 215

Ala Ala Ala Ser Ile Ser Gly Gly Ser Glu Ala Thir Trp Ala 225 23 O 235 24 O

Phe Glu Luell Thir Phe Thir Ser Thir Ala Wall Gly Lys Met 245 250 255

Wall Wall Glin Wall Thir Asn Thir Gly Ser Asp Luell Gly Ser Asn Thir Gly 26 O 265 27 O

Ala His Phe Asp Lell Glin Met Pro Gly Gly Gly Wall Gly Ile Asn 27s 285

Gly Cys Ala Thir Glin Trp Gly Ala Pro Thir Asp Gly Trp Gly Ala Arg 29 O 295 3 OO

Tyr Gly Gly Wall Ser Ser Ala Ser Asp Ser Asn Lell Pro Ser Ala 3. OS 310 315

Lell Glin Ala Gly Cys Trp Arg Phe Gly Trp Phe Asn Ala Asp 3.25 330 335

Asn Pro Thir Met Thir Glin Wall Thir Pro Ala Ile Thir 34 O 345 35. O

Ala Ser Gly Ser Arg Lys 355 360

<210s, SEQ ID NO 52 &211s LENGTH: 219 212. TYPE : PRT &213s ORGANISM: Mastotermes darwiniens is hindgut symbiont sp.

<4 OOs, SEQUENCE: 52

Met Phe Wall Ala Phe Wall Ile Gly Ala Luell Cys Asp Phe Ser Gly 1. 5 1O 15

Asn Gly Glin Thir Thir Arg Trp Asp Cys Pro Ser Ser 2O 25 3O

Trp Ser Lys Ala Glin Wall Ser His Wall Wall Asn Ser Asn Ala 35 4 O 45

Asn Asn Glin His Asp Ser Thir Wall Asp Luell Lys Ser Gly Asp Gly SO 55 6 O

Gly Pro Ser Ala Cys Ala Asp Glin Ala Pro Trp Ala Wall Asn Ser 65 70

Ser Phe Met Gly Thir Ala Ala Ala Ala Luell Ser Gly Ala Ser Glu 85 90 95

Ala Asp Luell Cys Phe Glu Luell Thir Phe Thir Ser Gly Thir 105 11 O

Pro Asn Gly Lys Met Lell Wall Glin Ile Thir Asn Thir Gly Ser Asp US 9.238,806 B2 119 120 - Continued

115 12 O 125

Lell Ser Gly Asn Glin Phe Asp Luell Luell Ile Pro Gly Gly Gly Wall Gly 13 O 135 14 O

Ile Phe Asp Gly Cys Thir Arg Glin Pro Gly Ser Tyr Asp Trp Gly 145 150 155 160

Glin Arg Tyr Gly Gly Wall Thir Ser Arg Asp Gly Ser Luell Pro 1.65 17O 17s

Ser Thir Luell Glin Thir Gly Glin Phe Arg Phe Asp Ile Gly Asp 18O 185 19 O

Asn Pro Ser Wall Ser Phe Ser Thir His Pro Asp Ser Ile Wall 195 2O5

Gly Lys Thir Asn Ser Arg Arg Asn Asp Asp Ala 21 O 215

SEO ID NO 53 LENGTH: 218 TYPE : PRT ORGANISM: Magnaporthe grisea

< 4 OOs SEQUENCE: 53

Met Lys Ile Thr Thir Ser Ala Wall Luell Ala Lell Thir Ala Ala Wall 1. 5 1O 15

Ser Ala Glin Wall Glin Gly Thir Gly Ala Thir Thir Arg Trp Asp 25

Pro Ser Cys Gly Trp Pro Gly Ala Asn Lell Ala Ser Gly 35 4 O 45

Pro Luell Arg Thir Cys Asp Lys Ala Asp Asn Pro Lell Asn Asp Gly Gly SO 55 6 O

Asn Thir Ser Gly Cys Asp Asn Gly Gly Gly Ala Phe Met Ser 65 70

Ser Glin Glu Pro Lell Ala Wall Asp Asp Ser Luell Ala Gly Phe Ala 85 90 95

Ala Wall Arg Ile Ser Gly Glin Arg Glu Ser Asp Trp Cys Ala 105 11 O

Glu Luell Thir Phe Thir Asn Luell Luell Arg ASn Thir Gly Gly Asp Luell 115 12 O 125

Gly Glin Asn His Phe Asp Ile Ala Met Pro Gly Gly Gly Wall Gly Ile 13 O 135 14 O

Phe Asn Ala Thir Glu Glin Tyr Gly Ala Pro Ala Asn Gly Trp Gly 145 150 155 160

Glu Arg Gly Gly Wall Arg Ser Arg Ser Glu Asp Ala Phe Pro 1.65 17O 17s

Glu Luell Lys Lys Gly Trp Arg Phe Asp Trp Phe Gly 18O 185 19 O

Ala Asp Asn Pro Ser Wall Ser Phe Glin Wall Thir Cys Pro Ser Glu 195 2OO 2O5

Lell Thir Ser Ser Gly Cys Wall Arg Ala 21 O 215

<210s, SEQ ID NO 54 &211s LENGTH: 219 212. TYPE : PRT &213s ORGANISM: Mastotermes darwiniens is hindgut symbiont sp.

<4 OOs, SEQUENCE: 54

Met Phe Wall Ala Phe Wall Ile Gly Ala Lieu. Cys Lys Asp Tyr Ser Gly US 9.238,806 B2 121 122 - Continued

1O 15

Asn Gly Glin Thir Thir Arg Trp Asp Cys Pro Ser Ser 2O 25 3O

Trp Ser Lys Ala Glin Wall Ser His Wall Wall Asn Ser Asn Ala 35 4 O 45

Asn Gly Glin His Asp Ser Thir Wall Asp Luell Lys Ser Gly Asp Gly SO 55 6 O

Gly Pro Ser Ala Cys Thir Asp Glin Ala Pro Trp Ala Wall Asn Ser 65 70

Ser Phe Met Gly Thir Ala Ala Ala Ala Luell Ser Gly Gly Ser Glu 85 90 95

Ser Asp Luell Cys Cys Phe Glu Luell Thir Phe Thir Ser Gly Thir 105 11 O

Pro Asn Gly Lys Lys Met Lell Wall Glin Ile Thir Asn Thir Gly Ser Asp 115 12 O 125

Lell Ser Gly Asn Glin Phe Asp Luell Luell Ile Pro Gly Gly Gly Wall Gly 13 O 135 14 O

Ile Phe Asp Gly Thir Arg Glin Pro Gly Ser Asp Trp Gly 145 150 155 160

Glin Arg Gly Gly Wall Thir Ser Arg Asp Gly Ser Luell Pro 1.65 17O 17s

Ser Ala Luell Glin Ser Gly Phe Glin Phe Arg Phe Asp Ile Ala Asp 18O 185 19 O

Asn Pro Ser Wall Ser Phe Ser Thir His Cys Pro Asp Thir Ile Wall 195 2O5

Ser Lys Pro Thir Arg Arg Asn Asp Asp Ser 21 O 215

<210s, SEQ ID NO 55 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Mastotermes darwiniens is hindgut symbiont sp.

<4 OOs, SEQUENCE: 55

Met Ile Wal Wall Phe Wall Ile Gly Ala Luell Cys Asp Ser Gly 1. 5 15

Asn Gly Glin Thir Thir Arg Trp Asp Cys Pro Ser 2O 25

Trp Ser Lys Ala Glin Wall Ser His Wall Wall Asn Ser Thir Ala 35 4 O 45

Thir Gly Ser His Asp Thir Thir Wall Asp Luell Lys Ser Gly Asp Gly SO 55 6 O

Gly Pro Ser Wall Cys Wall Asp Glin Ala Pro Trp Ala Wall Asn Ser 65 70 8O

Ser Phe Met Gly Thir Ala Ala Ala Ala Luell Ser Gly Gly Ser Glu 85 90 95

Ser Asp Luell Cys Cys Arg Phe Glu Luell Thir Phe Thir Ser Gly Glin 105 11 O

Pro Asn Gly Lys Lys Met Lell Wall Glin Wall Thir Asn Thir Gly Ser Asp 115 12 O 125

Lell Ser Gly Asn Glin Phe Asp Luell Luell Ile Pro Gly Gly Gly Wall Gly 13 O 135 14 O

Ile Phe Asp Gly Ser Arg Glin Pro Gly Gly Asn Asp Trp 145 150 155 160 US 9.238,806 B2 123 124 - Continued Gly Glin Arg Tyr Gly Gly Val Thir Ser Lys Ala Gly Cys Ala Lys Ile 1.65 17O 17s Pro Ala Glu Lieu Lys Ala Gly Cys Glu Phe Arg Phe Asp Tyr Ile Gly 18O 185 19 O Asp Asn Pro Ser Val Ser Phe Llys Ser Val His Cys Pro Asp Thir Ile 195 2OO 2O5 Thir Ser Lys Thir Asn. Cys Arg Arg Asn Asp Asp Asn 21 O 215 22O

<210s, SEQ ID NO 56 &211s LENGTH: 220 212. TYPE: PRT <213> ORGANISM: Mastotermes darwiniens is hindgut symbiont sp. <4 OOs, SEQUENCE: 56 Met Ile Val Val Phe Val Ile Gly Ala Lieu. Cys Lys Asp Tyr Ser Gly 1. 5 1O 15 Ser Gly Lys Thr Thr Arg Tyr Trp Asp Cys Cys Llys Pro Ser Cys Ser 2O 25 3O Trp Ser Lys Lys Ala Glin Val Ser His Val Val Asn. Ser Cys Thr Ala 35 4 O 45 Ser Gly Gln His Asp Thir Thr Val Asp Lieu Lys Ser Gly Cys Asp Gly SO 55 6 O Gly Pro Ser Tyr Val Cys Val Asp Glin Ala Pro Trp Ala Val Asin Ser 65 70 7s 8O Ser Tyr Phe Met Gly Thr Ala Ala Ala Ala Leu Ser Gly Gly Ser Glu 85 90 95 Ser Asp Lieu. Cys Cys Arg Cys Phe Glu Lieu. Thr Phe Thr Ser Gly Glin 1OO 105 11 O Ser Asn Gly Lys Llys Met Lieu Val Glin Ile Thr Asn Thr Gly Ser Asp 115 12 O 125 Lieu. Ser Gly Asn. Glin Phe Asp Lieu. Lieu. Ile Pro Gly Gly Gly Val Gly 13 O 135 14 O Ile Phe Asp Gly Cys Ser Arg Glin Tyr Pro Gly Gly Asn Tyr Asp Trp 145 150 155 160 Gly Glin Arg Tyr Gly Gly Val Thir Ser Lys Ala Gly Cys Ala Lys Ile 1.65 17O 17s Pro Ala Glu Lieu Lys Ala Gly Cys Glu Phe Arg Phe Asp Tyr Ile Gly 18O 185 19 O Asp Asn Pro Ser Val Ser Phe Llys Ser Val His Cys Pro Asp Thir Ile 195 2OO 2O5 Thir Ser Lys Thir Asn. Cys Arg Arg Asn Asp Asp Glin 21 O 215 22O

<210s, SEQ ID NO 57 &211s LENGTH: 614 212. TYPE: PRT <213> ORGANISM: Pichia pastoris

<4 OO > SEQUENCE: 57

Met Ser Thr Lieu. Thir Lieu. Lieu Ala Wall Lieu. Lieu. Ser Lieu. Glin Asn. Ser 1. 5 1O 15 Ala Lieu Ala Ala Glin Ala Glu Thir Ala Ser Lieu. Tyr His Glin Cys Gly 2O 25 3O

Gly Ala Asn Trp Glu Gly Ala Thr Glin Cys Ile Ser Gly Ala Tyr Cys 35 4 O 45 US 9.238,806 B2 125 126 - Continued

Glin Ser Glin Asn Pro Tyr Glin Wall Ala Thir Ser Trp Gly SO 55 6 O

Tyr Thir Asn Thir Ser Ile Ser Ser Thir Ala Thir Lell Pro Ser Ser 65 70

Ser Thir Thir Wall Ser Pro Thir Ser Ser Wall Wall Pro Thir Gly Luell Wall 85 90 95

Ser Pro Luell Tyr Gly Glin Gly Gly Glin ASn Trp Asn Gly Ala Thir 105 11 O

Ser Ala Glin Gly Ser Cys Met Asn Asn Tyr Phe 115 12 O 125

Glin Cys Wall Pro Glu Ala Asp Gly Asn Pro Ala Glu Ile Ser Thir Phe 13 O 135 14 O

Ser Glu Asn Gly Glu Ile Ile Wall Thir Ala Ile Glu Ala Pro Thir Trp 145 150 155 160

Ala Glin Gly Gly His Gly Tyr Gly Pro Thir Glin Wall 1.65 17O 17s

Gly Thir Ser Cys Arg Glu Lell Asn Ala Trp Glin Cys Ile Pro 185 19 O

Asp Asp His Thir Asp Ala Ser Thir Thir Thir Luell Asp Pro Thir Ser Ser 195

Phe Wall Ser Thir Thir Ser Lell Ser Thir Luell Pro Ala Ser Ser Glu Thir 21 O 215

Thir Ile Wall Thir Pro Thir Ser Ile Ala Ala Glu Glin Wall Pro Luell Trp 225 23 O 235 24 O

Gly Gln Gly Gly Ile Gly Tyr Thr Gly Ser Thr Ile Glu Gln 245 250 255

Gly Ser Wall Tyr Lell Asn Asp Trp Glin Luell Ile Ser 26 O 265 27 O

Asp Glin Gly Thir Ala Ser Thir Thir Ser Ala Thir Thir Ser Ile Thir Ser 27s 285

Phe Asn Wall Ser Ser Ser Ser Glu Thir Thir Wall Ile Ala Pro Thir Ser 29 O 295 3 OO

Ile Ser Thir Glu Asp Wall Pro Luell Trp Gly Glin Gly Gly Ile Gly 3. OS 310 315

Thir Gly Ser Thir Thir Ser Glin Gly Ser Wall Luell Asn 3.25 330 335

Asp Trp Phe Glin Lell Pro Glu Glu Glu Thir Thir Ser Ser Thir 34 O 345 35. O

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Thir Ser Ser Ala Ser Ser 355 360 365

Thir Ser Ser Thir Ser Ser Thir Ser Ser Thir Ser Ser Thir Ser Ser Ser 37 O 375 38O

Thir Ser Ser Ser Ser Ile Pro Thir Ser Thir Ser Ser Ser Gly Asp Phe 385 390 395 4 OO

Glu Thir Ile Pro Asn Gly Phe Ser Gly Thir Gly Arg Thir Thir Arg Tyr 4 OS 415

Trp Asp Cys Lys Pro Ser Ser Trp Pro Gly Ser Asn Ser 425 43 O

Wall Thir Gly Pro Wall Arg Ser Cys Gly Wall Ser Gly Asn Wall Luell Asp 435 44 O 445

Ala Asn Ala Glin Ser Gly Cys Ile Gly Gly Glu Ala Phe Thir Asp 450 45.5 460

Glu Glin Glin Pro Trp Ser Ile Asn Asp Asp Luell Ala Gly Phe Ala US 9.238,806 B2 127 128 - Continued

465 470

Ala Ala Ser Luell Ala Gly Gly Ser Glu Asp Ser Ser Thir Cys 485 490 495

Met Lel Thir Phe Thir Ser Ser Ser Ile Ala Gly Thir Met Ile SOO 505

Wall Glin Lel Thir Asn Thir Gly Ala Asp Luell Gly Ser Asn His Phe Asp 515 525

Ile Ala Lel Pro Gly Gly Gly Luell Gly Ile Phe Thir Glu Gly Ser 53 O 535 54 O

Ser Glin Phe Gly Ser Gly Glin Trp Gly ASn Glin Gly Gly Ile 5.45 550 555 560

Ser Ser Lel Ala Glu Asp Gly Luell Pro Ser Glu Lell Glin Pro Gly 565 st O sts

Glin Phe Arg Phe Gly Trp Phe Glu Asn Ala Asp Asn Pro Ser Wall 585 59 O

Glu Phe Glu Glin Wall Ser Pro Pro Glu Ile Thir Ser Ile Thir Gly 595 6OO 605

Ala Arg Thir Asp Glu 610

<210s, SEQ ID NO 58 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Piromyces equi

<4 OOs, SEQUENCE: 58

Met Arg Lieu Ala Lell Thir Ser Ile Ala Luell Ala Ala Ser Ile Ala 1. 5 1O 15

Lys Wall Ser Ala Ala Trp Ala Glin Ser Glin Gly Asn Cys 2O 25 3O

Asn Asn Pro Ser Ser Thir Wall Glu Thir Asp Ser Glin 35 4 O

Trp Gly Wall Glin Asn Gly Glin Trp Gly Ile Asp Ser Gly SO 55 6 O

Glin Asn Glin Gly Asn Glu Ser Thir Gly ASn Gly Ser Pro Cys 65 70

Asn Thir Glin Ala Thir Thir Asp Gly Asp Gly Asp Trp Ala 85 90 95

Phe Glu Asn Gly Asn Trp Gly Ile ASn Ser Lys Glin Glin 1OO 105 11 O

Pro Glin Asn Asn Asn Glin Thir Gly Asn Gly Ala Tyr Arg Cys 115 12 O 125

Asn Thir Glin Ala Thir Tyr Thir Asp Asn Glu Gly Trp Ala Phe 13 O 135 14 O

Glu Asn Gly Asp Trp Cys Gly Ile Ser Pro Ser Glin Glin 145 150 155 160

Wall Thir Thir Thir Thir Thir Arg Arg Thir Thir Thir Thir Thir Glin Glin Glin 1.65 17O 17s

Glin Pro Thir Gly Ser Gly Gly Asn Ser Asn Wall Pro Lell Asn Pro Pro 18O 185 19 O

Asp Phe Ser Gly Glin Thir Gly Lys Thir Thir Arg Trp Asp Cys 195

Lell Ala Ser Ser Trp Glin Glu Asn Asn Asp Gly Ala Glin 21 O 215 22O US 9.238,806 B2 129 130 - Continued

Gly Wall Wall Arg Ser Cys Asn Wall Asp Gly Ile Thir Pro Phe Thir Asp 225 23 O 235 24 O

Lell Ser Asn Luell Trp Arg Wall Ser Gly Asn Gly Gly Ser Wall 245 250 255

Met Asn Asp Glin Glin Pro Trp Ala Ile Asn Asp Asn Wall Ala 26 O 265 27 O

Gly Phe Wall Ala Ser His Glu Thir Cys Glin Arg Luell 27s 28O 285

Phe Thir Ser Gly Pro Ile Ala Gly Glin Met Ile Wall Glin Thir 29 O 295 3 OO

Thir Asn Thir Gly Gly Asp Lell Ser Ser Asn His Phe Asp Ile Glin Met 3. OS 310 315

Pro Gly Gly Gly Phe Gly Ile Phe Asp Gly Thir Ser Glin Phe Gly 3.25 330 335

Gly Ser Glin Trp Gly Glu Arg Tyr Gly Gly Ile Ser Ser Ala Ser 34 O 345 35. O

Glin Ala Asn Lell Pro Pro Glin Luell Ala Gly Cys Glu Trp Arg 355 360 365

Phe Asn Trp Phe Lys Asn Ala Asp Asn Pro Ala Wall Wall Phe Glu Arg 37 O 375

Wall Glin Pro Lys Glu Lell Thir Glu Ile Thir Gly Wall Pro Gly 385 390 395 4 OO

Asp Asp Ala Ser Ala Luell Pro Trp 4 OS 41O

SEO ID NO 59 LENGTH: 237 TYPE : PRT ORGANISM: Apriona germari

< 4 OOs SEQUENCE: 59

Met Llys Val Phe Wall Ala Ile Luell Ala Wall Phe Thir Phe Glu Wall 1. 5 15

Ser Luell Ser Lys Wall Asn Luell Asn Wall Pro Gly Ile Ser 25

Gly Ser Gly Thir Thir Thir Arg Tyr Trp Asp Lys Pro Ser 35 4 O 45

Gly Trp Wall Glu Asn Lell Ala Glu Gly Thir Pro Wall Ala Thir SO 55 6 O

Ser Ala Asp Gly Ser Thir Thir Wall Ala Ala Ser Wall Ser Ser Cys 65 70

Wall Gly Gly Thir Ser Met Ser Asn Glin Glin Pro Ser Wall 85 90 95

Asn Ser Thir Phe Ala Lell Gly Phe Wall Ala Ala Ser Phe Thir Gly Gly 105 11 O

Ala Asp Thir Asn Tyr Ala Ile Lys Lell Thir Phe Glin Asp 115 12 O 125

Ala Luell Glin Gly Lys Thir Met Wall Wall Glin Wall Thir Asn Thir Gly Gly 13 O 135 14 O

Asp Luell Gly Ser Asn Glin Phe Asp Ile Ala Ile Pro Gly Gly Gly Wall 145 150 155 160

Gly Ile Phe Thir Asp Gly Ser Ser Glin Trp Gly Thir Pro Ser Asn 1.65 17O 17s

Gly Trp Gly Asp Glin Trp Trp Wall Trp Gly Ser Glu Ala Asp 18O 185 19 O US 9.238,806 B2 131 132 - Continued

Ala Glin Lieu Pro Ser Asp Lieu. Glin Glu Gly Cys Llys Phe Arg Phe Glu 195 2O5

Phe Met Glu Gly Ala Ser Asn Pro Gly Val Thr Phe Glu Glin Wall Asp 21 O 215 22O

Cys Pro Ser Glu Lieu Wall Ser Ile Thr Gly Cys Asn Tyr 225 23 O 235

SEQ ID NO 60 LENGTH: 239 TYPE : PRT ORGANISM: Apriona germari

< 4 OOs SEQUENCE: 6 O

Met Llys Val Lieu Lell Ala Wall Wall Ala Wall Lieu. Cys Thir Phe Glu Ala 1. 5 15

Ser Luell Ser Glin Asp His Wall Thir Pro Lieu. Wall Gly Gly Val Ser 25 3O

Gly Thir Gly Thir Thir Arg Tyr Trp Asp Lys Pro Ser 35 4 O 45

Ser Trp Ala Asn Lell Lys Ser Ser Gly Lys Pro Wall Glu Ala SO 55 6 O

Cys Ala Ala Asp Gly Lys Thir Wall Wall Lys Glu Ser Thir Llys Ser Ala 65 70 7s

Glu Glu Gly Ala Gly Ala Met Cys Ser Asp Glin Gln Pro 85 90 95

Wall Wall Asn Ser Thr Phe Ala Lieu Gly Tyr Wall Ala Ala Ser Phe Thr 105 11 O

Gly Gly Ile Asp Wall Asn Met Cys Ala Cys Lieu Arg Lieu Lys Phe 115 12 O 125

Glin Gly Asp Luell Ser Gly Lys Glin Met Ile Wall Glin Wall Thir Asn Thir 13 O 135 14 O

Gly Ser Asp Luell Gly Ser Asn Glin Phe Asp Ile Ala Ile Pro Gly Gly 145 150 155 160

Gly Wall Gly Ile Phe Thir Gly Ser Ser Glin Trp Gly Thr Pro 1.65 17O 17s

Ser Asn Gly Trp Gly Asp Glin Tyr Gly Gly Wall Ser Ser Glu Ser Glin 18O 185 19 O

Ser Glin Luell Pro Ser Ser Luell Arg Glu Gly Cys Lys Phe Arg Phe 195

Thir Phe Met Ser Wall Ser Asn Pro Ala Wall. Thir Phe Glu Glin Wall 21 O 215

Ser Pro Ser Glu Ile Wall Ser Ala Ser Gly Cys Asn Tyr Ser 225 23 O 235

<210s, SEQ ID NO 61 &211s LENGTH: 271 212. TYPE : PRT <213> ORGANISM: Alternaria alternata

<4 OOs, SEQUENCE: 61

Met Pro Gln His Lieu. Arg Asn. Ile Ala Lieu. Thir Ile Glu Phe Phe Ala 1. 5 15

Val Lieu Ala Arg Cys Ala His Lieu. Asn Tyr Thr Gly Glu Ala Wall Thir 25 3O

Thr Arg Phe Trp Asp Cys Cys Llys Pro Ser Cys Gly Trp Asin Gly Lys 35 4 O 45 US 9.238,806 B2 133 134 - Continued

Ala Glin Phe Ser Arg Pro Wall Glu Ser Thir Ala Asp Asp Lys Pro SO 55 6 O

Thir Asp Ile Ala Ala Gly Thir Gly Asn Ser Gly Ser Ala Phe Glin 65 70

Ser Asn Glin Glin Pro Trp Ala Ile Asn Asp Thir Lell Ser Tyr Gly 85 90 95

Ala Gly Wall Tyr Ile Thir Pro Asp Luell Thir His Gly Gly Ile Glu 1OO 105 11 O

Asp Ala Trp Cys Ala Tyr Glin Luell ASn Phe Thir Ser Glu Pro 115 12 O 125

Lell Ile Gly Ser Met Ile Wall Glin Ala Ser Asn Thir Ala Asp 13 O 135 14 O

Wall Thir Asn Ala Asn Arg Phe Ser Luell Ala Wall Pro Gly Gly Asn Thir 145 150 155 160

Thir Ser Thir Asn Ala Ala Glin Glin Tyr Gly Wall Ser Glin Ser Wall 1.65 17O 17s

Phe Gly Asn Met Ala Gly Wall Lys Ser Ile Asp Asp Cys Glin Asn 18O 185 19 O

Lell Pro Asn Lell Arg Ala Gly Cys Glu Trp Arg Phe Asp Trp Phe

Asn Ser Phe Pro Ser Ala Asn Phe Arg Wall Wall Pro 21 O 215

Ser Glu Thir Ala Lys Thir Asn Ile Arg Asn Asp Asp Wall 225 23 O 235 24 O

Lell Ala Glu Ala Ser Ser Ala Glin Ser Luell Thir Pro Ser Ser Ser 245 250 255

Thir Met Phe Phe Ala Wall Ile Ile Luell Gly Lell Ile Ser Ile 26 O 265 27 O

<210s, SEQ ID NO 62 &211s LENGTH: 242 212. TYPE : PRT &213s ORGANISM: Phaedon cochleariae

<4 OOs, SEQUENCE: 62

Met Glin Wall Ile Wall Lell Pro Luell Wall Phe Luell Ala Thir Phe Ala Thir 1. 5 15

Ser Gly Ser Luell Ala Ala Pro Asp Ala Ser Pro Glu Ile Wall Pro Wall 25

Asp Gly Gly Luell Ser Gly Gly Thir Thir Thir Arg Tyr Trp Asp 35 4 O 45

Lys Pro Ser Ala Trp Glu Asn Ile Asn Thir Pro Thir Met SO 55 6 O

Thir Pro Wall Glin Thir Cys Ala Ile Asp Gly ASn Thir Wall Wall Asn Ala 65 70

Ser Wall Glin Ser Gly Ile Gly Gly Ser Ser Met Ser Asn 85 90 95

Glin Glin Ala Phe Wall Wall Asn Ser Thir Luell Ala Phe Gly Phe Ala Ala 105 11 O

Gly Ser Phe Thir Gly Gly Wall Asp Asn Asn Luell Cys Ser Met 115 12 O 125

Lell Luell Thir Phe Glin Gly Glin Luell Ala Gly Lys Glin Phe Luell Wall Glin 13 O 135 14 O

Ile Thir Asn Thir Gly Gly Asp Luell Gly Ser Thir Ser Ser Ile Trp Pro