Novel Cellulases and Their Uses

• US 20100204080A1
• Filed: 04/15/2010
• Published: 08/12/2010
• 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: Paloheimo, Marja, Ojapalo, Pentti, Alapuranen, Marika, Puranen, Terhi, Valtakari, Leena, Kallio, Jarno, Vehmaanperä, Jari
• Granted Patent: US 7,981,654 B2
• Time in Patent Office: Days
• US Class Current: 510/392
• 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:

an optionally modified first amino acid sequence of a cellulase core derived from one species; and

an optionally modified second amino acid sequence of a linker and cellulose binding domain (CBD) derived from another species, 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 first amino acid sequence is from a neutral cellulase and said second amino acid sequence is from an acid cellulase.

5. The cellulase fusion protein of claim 4, wherein said first amino acid sequence is from a cellulase of family 45 and said second amino acid sequence is from a cellulase of family 7.

6. The cellulase fusion protein of claim 4, wherein said neutral cellulase is of fungal origin.

7. The cellulase fusion protein of claim 6, wherein said neutral cellulase is derived from a genus selected from the group consisting of Melanocarpus, Humicola, Thielavia, Myceliophthora, Fusarium, Acremonium, Chrysosporium, Thermoascus, Scopulariopsis, Myriococcum, Talaromyces and Chaetomium.

8. The cellulase fusion protein of claim 7, wherein said cellulase is Melanocarpus albomyces 20 K cellulase, 50K cellulase, 50KB cellulase, or a derivative thereof.

9. The cellulase fusion protein of claim 7, wherein said cellulase is Thermoascus aurantiacus CBHI or a derivative thereof.

10. The cellulase fusion protein of claim 4, wherein said acid cellulase is derived from Trichoderma sp. or Hypocrea.

11. The cellulase fusion protein of claim 1, wherein

the first amino acid sequence is the 20 K cellulase of Melanocarpus albomyces of SEQ ID NO:

2, optionally 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

the second amino acid sequence is the linker and CBD of Trichoderma reesei cellobiohydrolase I of SEQ ID NO;

4, optionally 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.

12. The cellulase fusion protein of claim 11, wherein said second amino acid sequence is set forth as SEQ ID NO:

44.

13. 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-³Ile-⁴Pro-⁵Ser-⁶Ser.

14. The cellulase fusion protein of claim 11, 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.

15. The cellulase fusion protein of claim 14, wherein said second amino acid sequence is selected from a group of SEQ ID. NO:

45, 46, 47, and 48.

16. The cellulase fusion protein of claim 11, 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.

17. The cellulase fusion protein of claim 16, wherein said second amino acid sequence is selected from the group of SEQ ID. NO:

49 and 50.

18. The cellulase fusion protein of claim 11, 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.

19. The cellulase fusion protein of claim 11, 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.

20. The cellulase fusion protein of claim 11, 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.

21. The cellulase fusion protein of claim 11, 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.

22. The cellulase fusion protein of claim 11, wherein said first amino acid sequence is selected from the group of SEQ ID. NO:

37, 38, 39, and 40.

23. A process for the production of a cellulase fusion protein of claim 1, comprising the steps of culturing a host cell comprising an expression vector comprising

a first polynucleotide encoding a an optionally modified first amino acid sequence of a cellulase core derived from one species;

and

a second polynucleotide encoding an optionally modified second amino acid sequence of a linker and cellulose binding domain (CBD) derived from another species, and

a polynucleotide encoding a junction region connecting said first and second amino acid sequence and having the following general formula;

24. The process of claim 23, wherein the first polynucleotide encodes a first amino acid sequence of 20 K cellulase of Melanocarpus albomyces of SEQ ID NO:

2, optionally 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

the second polynucleotide encodes a second amino acid sequence of the linker and CBD of Trichoderma reesei cellobiohydrolase I of SEQ ID NO;

4, optionally 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.

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

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

27. The process of claim 26, wherein the fabric or garment is denim.

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

29. The process of claim 28, wherein the textile material is manufactured of natural cellulose containing fibers or manmade cellulose containing fibers or a mixture thereof.

30. The process of claim 28, wherein the textile material is a blend of synthetic fibers and cellulose containing fibers.

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

32. A method of treating cellulosic fiber containing textile material, wherein said method comprises contacting said textile material with the detergent composition of claim 31.

33. 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.

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

35. The process for biofinishing of claim 28, herein said textile material is fabric, garment or yarn.

36. The detergent composition of claim 31, wherein said auxiliaries are selected from the group consisting of surface active agents, surfactants, bleaching agents and builders.