Cellulases and their uses

• US 7,981,654 B2
• Filed: 04/15/2010
• Issued: 07/19/2011
• Est. Priority Date: 04/29/2005
• Status: Active Grant

• Current Assignee: AB Enzymes Oy (Garfield Weston Foundation)
• Original Assignee: AB Enzymes Oy (Garfield Weston Foundation)
• Inventors: Alapuranen, Marika, Puranen, Terhi, Valtakari, Leena, Kallio, Jarno, Paloheimo, Marja, Ojapalo, Pentti, Vehmaanperä, Jari
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US12/760,744
• Publication Number:
  US 20100204080A1
• Time in Patent Office: 460 Days
• Field of Search: 435/209, 435/183, 435/262, 435/263, 510/114, 510/300, 510/374, 510/392, 510/531, 510/535, 536/23.4, 536/23.7, 811/551, 811/61
• US Class Current: 435/209
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01091 Cellulose 1,4-beta-cellobiosidase (3.2.1.91)

Abstract

The present invention provides novel cellulase fusion proteins, preparations of cellulase fusion proteins and compositions of cellulase fusion proteins. The present invention further provides cellulase expression vectors, host cells expressing cellulase and methods for preparing such vectors and cells. Uses of cellulases, cellulase preparations and cellulase compositions in the textile, detergent, pulp and paper industries are also provided.

Claims

1. A cellulase fusion protein comprising:

first amino acid sequence, which is the 20 K cellulase of Melanocarpus albomyces of SEQ ID NO;

2, or the 20K cellulase of Melanocarpus albomyces of SEQ ID NO;

2 having at least one modification selected from the group consisting of;

deletion of Ala at position 207, deletion of Val at position 208, substitution of Phe209Trp, and insertion of Pro after position 206, or a fragment thereof having cellulase activity, and

a second amino acid sequence, which is the linker and cellulose binding domain (CBD) of Trichoderma reesei cellobiohydrolase I of SEQ ID NO;

4, or the linker and cellulose biding domain (CBD) of Trichoderma reesei cellobiohydrolase I of SEQ ID NO;

4 having at least one modification selected from the group consisting of;

substitution of tyrosine at position 492 (position 31 of the CBD), 493 (position 32 of the CBD) of the mature T. reesei CBHI with an aliphatic or aromatic amino acid, and deletions of amino acids 434 to 444 or 434 to 460 of the mature T. reesei CBHI sequence, or a fragment thereof having CBD activity, and

a junction region having the following general formula;

2. The cellulase fusion protein of claim 1, wherein the junction region has the following formula:

3. The cellulase fusion protein of claim 1, wherein the junction region has the following formula:

4. The cellulase fusion protein of claim 1, wherein said second amino acid sequence is set forth as SEQ ID NO:

44.

5. The cellulase fusion protein of claim 1, wherein said first amino acid sequence is set forth as SEQ ID NO:

39, said second amino acid sequence is set forth as SEQ ID NO;

44, and said junction region has the formula;

¹Val-²Gln-³lle-⁴Pro-⁵Ser-⁶Ser (SEQ ID NO;

     52).

6. The cellulase fusion protein of claim 1, wherein in said second amino acid sequence at least one of said tyrosine at position 492 (position 31 of the CBD) and 493 (position 32 of the CBD) of the mature T. reesei CBHI is substituted with alanine or tryptophan.

7. The cellulase fusion protein of claim 1, wherein in said second amino acid sequence amino acids 434 to 444 or 434 to 460 of the mature T reesei CBHI sequence are deleted.

8. The cellulase fusion protein of claim 1, wherein in said first amino acid sequence valine at position 208 of the 20K cellulase sequence of M albomyces of SEQ ID. NO:

2 has been deleted.

9. The cellulase fusion protein of claim 1, wherein in said first amino acid sequence alanine at position 207 of the 20K cellulase sequence of M albomyces of SEQ ID. NO:

2 has been deleted.

10. The cellulase fusion protein of claim 1, wherein in said first amino acid sequence phenylalanine at position 209 of the 20 K cellulase sequence of M albomyces of SEQ ID. NO:

2 has been substituted with Trp.

11. The cellulase fusion protein of claim 1, wherein said first amino acid sequence contains an inserted proline after position 206 in the 20 K cellulase sequence of M albomyces of SEQ ID. NO:

2.

12. The cellulase fusion protein of claim 1, wherein said first amino acid sequence is SEQ ID. NO:

39.

13. An enzyme preparation comprising the cellulase fusion protein of claim 1.

14. A process for biostoning which comprises the step of adding the cellulase fusion protein of claim 1 to cotton containing fabric or garments.

15. The process of claim 14, wherein the fabric or garment is denim.

16. A process for biofinishing, which comprises the step of adding the cellulase fusion protein of claim 1 to a textile material.

17. The process of claim 16, wherein the textile material is manufactured of natural cellulose containing fibers or manmade cellulose containing fibers or a mixture thereof.

18. The process of claim 16, wherein the textile material is a blend of synthetic fibers and cellulose containing fibers.

19. A detergent composition comprising the fusion protein of claim 1 and one or more auxiliaries.

20. A method of treating cellulosic fiber containing textile material, wherein said method comprises contacting said textile material with the detergent composition of claim 19.

21. A method for treating wood-derived pulp or fiber, which comprises the step of adding the cellulase fusion protein of claim 1 to wood-derived mechanical or chemical pulp or secondary fiber.

22. A method for improving the quality of animal feed, which comprises treating plant material with the cellulase fusion protein of claim 1.

23. The process for biofinishing of claim 16, herein said textile material is fabric, garment or yarn.

24. The detergent composition of claim 19, wherein said auxiliaries are selected from the group consisting of surface active agents, surfactants, bleaching agents and builders.