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(12) United States Patent (10) Patent No.: US 9,187,739 B2 Lange Et Al

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(12) United States Patent (10) Patent No.: US 9,187,739 B2 Lange Et Al US009 187739B2 (12) United States Patent (10) Patent No.: US 9,187,739 B2 Lange et al. (45) Date of Patent: Nov. 17, 2015 (54) POLYPEPTIDES HAVING CI2N 9/42 (2013.01); CI2P 7/14 (2013.01); CELLOBO HYDROLASE ACTIVITY AND CI2Y 302/01091 (2013.01); Y02E 50/17 POLYNUCLEOTDESENCODING SAME (2013.01) (58) Field of Classification Search (71) Applicant: Novozymes Aktieselskab. Bagsvaerd CPC .......... C12N 1/00; C12N 1/22; C12N 9/2405: (DK) C12N 9/2408; C12N 9/2334; C12N 9/2437; C12N 9/2445; C12N 15/70; C12N 15/74; (72) Inventors: Lene Lange, Valby (DK); Wenping Wu, C12N 15779 Beijing (CN); Dominique Aubert, See application file for complete search history. Copenhagen (DK); Sara Landvik, Holte (DK); Kirk Matthew Schnorr, Holte (56) References Cited (DK); Ib Groth Clausen, Birkeroed (DK) U.S. PATENT DOCUMENTS 5, 120,463 A 6/1992 Bjork et al. (73) Assignee: Novozymes A/S, Bagsvaerd (DK) 5.487,989 A 1/1996 Fowler et al. 5,955,270 A 9, 1999 Radford et al. (*) Notice: Subject to any disclaimer, the term of this 6,184,019 B1 2/2001 Miettinen-Oinonen et al. patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS WO 95,02675 A1 1, 1995 (21) Appl. No.: 14/668,532 WO 97.14804 A1 4f1997 WO 99.06574 A1 2, 1999 (22) Filed: Mar. 25, 2015 WO 01.04284 A1 1, 2001 WO O1,25468 A1 4/2001 (65) Prior Publication Data WO O1,79507 A2 10, 2001 US 2015/0218542 A1 Aug. 6, 2015 OTHER PUBLICATIONS Azevedo et al., Nucleic Acids Research, vol. 18, No.3, p. 668 (1990). Emalfarb, Geneseq Accession No. ABA92722 (2002). Related U.S. Application Data Gams et al., Transacations British Mycological Society, vol. 59, No. (60) Continuation of application No. 14/064,398, filed on 3, pp. 519-522 (1972). Oct. 28, 2013, now Pat. No. 8,993,299, which is a Ganju et al., Biochimica et Biophysica Acta, vol. 993, pp. 266-274 (1989). division of application No. 13/681,490, filed on Nov. Gielkens et al., Applied and Environmental Microbiology, vol. 65. 20, 2012, now Pat. No. 8,603,794, which is a division No. 10, pp. 4340-4345 (1999). of application No. 13/646,980, filed on Oct. 8, 2012, Hong et al., EBI Accession No. AF421954 (2001). now Pat. No. 8,507,238, which is a division of Hong et al., EBI Accession No. Q96UR5 (2001). application No. 13/483,389, filed on May 30, 2012, Hong et al., EBI Accession No. AF478686 (2002). now Pat. No. 8,603.793, which is a division of Hong et al., Uniprot Accession No. Q8TG37 (2002). application No. 12/818,861, filed on Jun. 18, 2010, Kvachadze et al., Biosis Accession No. PREV 1998.00120135 (1997). Kvachadze et al., Mikrobiologiya, vol. 66, No. 5, pp. 644-649 (1997). now Pat. No. 8.338,156, which is a continuation of Li et al., Journal of Applied Microbiology, vol. 106, pp. 1867-1875 application No. 10/481,179, filed as application No. (2009). PCT/DK02/00429 on Jun. 26, 2002, now Pat. No. Radford et al., EMBL Accession No. X17258 (1990). 7,785,853. Riske et al., Applied and Environmental Microbiology, vol. 56, No. 11, pp. 3261-3265 (1990). (30) Foreign Application Priority Data Takada et al., Journal of Fermentation and Bioengineering, vol. 85, No. 1 pp. 1-9 (1998). Jun. 26, 2001 (DK) ................................. 2001 O1OOO Taleb et al., Gene, vol. 161, pp. 137-138 (1995). Azevedo et al., Journal of General Microbiology, vol. 136, pp. 2569 (51) Int. Cl. 2576 (1990). CI2P 7/06 (2006.01) CI2P 7/08 (2006.01) Primary Examiner — Jana Hines CI2P 7/10 (2006.01) (74) Attorney, Agent, or Firm — Eric J. Fechter CI2P 7/14 (2006.01) CI2P 19/34 (2006.01) (57) ABSTRACT CI2N 9/26 (2006.01) The present invention relates to polypeptides having cello CID 3/386 (2006.01) biohydrolase I activity and polynucleotides having a nucle CI2N 9/42 (2006.01) otide sequence which encodes for the polypeptides. The CI2N 9/24 (2006.01) invention also relates to nucleic acid constructs, vectors, and (52) U.S. Cl. host cells comprising the nucleic acid constructs as well as CPC .............. CI2N 9/2408 (2013.01); CI ID3/386 methods for producing and using the polypeptides. (2013.01); CIID3/38645 (2013.01); CI2N 9/2437 (2013.01); C12N 9/2477 (2013.01); 23 Claims, No Drawings US 9, 187,739 B2 1. 2 POLYPEPTDES HAVING SUMMARY OF THE INVENTION CELLOBOHYDROLASE ACTIVITY AND POLYNUCLEOTDESENCODING SAME In a first aspect the present invention relates to a polypep tide having cellobiohydrolase I activity, selected from the CROSS-REFERENCE TO RELATED group consisting of: APPLICATIONS (a) a polypeptide comprising an amino acid sequence Selected from the group consisting of: This application is a continuation of U.S. application Ser. an amino acid sequence which has at least 80% identity No. 14/064,398 filed on Oct. 28, 2013, now U.S. Pat. No. with amino acids 1 to 526 of SEQID NO:2, 8,993,299, which is a divisional of U.S. application Ser. No. 10 an amino acid sequence which has at least 80% identity 13/681,490 filed on Nov. 20, 2012, now U.S. Pat. No. 8,603, with amino acids 1 to 529 of SEQID NO:4, 794, which is a divisional of U.S. application Ser. No. 13/646, an amino acid sequence which has at least 80% identity 980 filed on Oct. 8, 2012, now U.S. Pat. No. 8,507,238, which with amino acids 1 to 451 of SEQID NO:6, is a divisional of U.S. application Ser. No. 13/483,389 filed on an amino acid sequence which has at least 80% identity May 30, 2012, now U.S. Pat. No. 8,603.793, which is a 15 with amino acids 1 to 457 of SEQID NO:8, divisional of U.S. application Ser. No. 12/818,861 filed on an amino acid sequence which has at least 80% identity Jun. 18, 2010, now U.S. Pat. No. 8.338,156, which is a con with amino acids 1 to 538 of SEQID NO:10, tinuation of U.S. application Ser. No. 10/481,179 filed Dec. an amino acid sequence which has at least 70% identity 17, 2003, now U.S. Pat. No. 7,785,853, which is a 35 U.S.C. with amino acids 1 to 415 of SEQID NO:12, 371 national application of international application no. PCT/ an amino acid sequence which has at least 70% identity DK02/000429 filed Jun. 26, 2002, which claims priority or with amino acids 1 to 447 of SEQID NO:14, the benefit under 35 U.S.C. 119 of Danish application no. PA an amino acid sequence which has at least 80% identity 2001 01000 filed on Jun. 26, 2001. The contents of these with amino acids 1 to 452 of SEQID NO:16, applications are fully incorporated herein by reference. an amino acid sequence which has at least 80% identity 25 with amino acids 1 to 454 of SEQID NO:38, FIELD OF THE INVENTION an amino acid sequence which has at least 80% identity with amino acids 1 to 458 of SEQID NO:40, The present invention relates to polypeptides having cel an amino acid sequence which has at least 80% identity lobiohydrolase I (also referred to as CBH I or CBH 1) activity with amino acids 1 to 450 of SEQID NO:42, and polynucleotides having a nucleotide sequence which 30 an amino acid sequence which has at least 80% identity encodes for the polypeptides. The invention also relates to with amino acids 1 to 446 of SEQID NO:44, nucleic acid constructs, vectors, and host cells comprising the an amino acid sequence which has at least 80% identity nucleic acid constructs as well as methods for producing and with amino acids 1 to 527 of SEQID NO:46, using the polypeptides. an amino acid sequence which has at least 80% identity 35 with amino acids 1 to 455 of SEQID NO:48, BACKGROUND OF THE INVENTION an amino acid sequence which has at least 80% identity with amino acids 1 to 464 of SEQID NO:50, Cellulose is an important industrial raw material and a an amino acid sequence which has at least 80% identity Source of renewable energy. The physical structure and mor with amino acids 1 to 460 of SEQID NO:52, phology of native cellulose are complex and the fine details of 40 an amino acid sequence which has at least 80% identity its structure have been difficult to determine experimentally. with amino acids 1 to 450 of SEQID NO:54, However, the chemical composition of cellulose is simple, an amino acid sequence which has at least 80% identity consisting of D-glucose residues linked by beta-1,4-glyco with amino acids 1 to 532 of SEQID NO:56, sidic bonds to form linear polymers with chains length of over an amino acid sequence which has at least 80% identity 10,000 glycosidic residues. 45 with amino acids 1 to 460 of SEQID NO:58, In order to be efficient, the digestion of cellulose requires an amino acid sequence which has at least 80% identity several types of enzymes acting cooperatively. At least three with amino acids 1 to 525 of SEQID NO:60, and categories of enzymes are necessary to convert cellulose into an amino acid sequence which has at least 80% identity glucose: endo(1,4)-beta-D-glucanases (EC 3.2.1.4) that cut with amino acids 1 to 456 of SEQID NO:66: the cellulose chains at random; cellobiohydrolases (EC 50 (b) a polypeptide comprising an amino acid sequence 3.2.1.91) which cleave cellobiosyl units from the cellulose selected from the group consisting of: chain ends and beta-glucosidases (EC 3.2.1.21) that convert an amino acid sequence which has at least 80% identity cellobiose and soluble cellodextrins into glucose.
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