Novel cellulases and their uses

• US 20060246566A1
• Filed: 04/29/2005
• Published: 11/02/2006
• Est. Priority Date: 04/29/2005
• Status: Active Grant

• Current Assignee: AB Enzymes Oy (Garfield Weston Foundation)
• Original Assignee: AB Enzymes GmbH (Garfield Weston Foundation)
• Inventors: Paloheimo, Marja, Ojapalo, Pentti, Alapuranen, Marika, Puranen, Terhi, Valtakari, Leena, Kallio, Jarno, Vehmaanpera, Jari
• Granted Patent: US 7,741,093 B2
• Time in Patent Office: Days
• US Class Current: 435/183
• 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/or cellulose binding domain (CBD) derived from another species, wherein a junction region has been introduced between said first amino acid sequence and said second amino acid sequence, whereby a stable fusion protein is obtained.

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

¹A-²B-³C-⁴D-⁵E-⁶F

wherein

¹A is selected from the group consisting of Gly, Ala, Leu, Pro, Ile, and Val;

²B is selected from the group consisting of Gly, Ala, Leu, Pro, Ile, Phe, Val, Glu, Asp, Gln, and Asn;

³C is selected from the group consisting of Gly, Ala, Lys, Leu, Pro, Ile, Val, Ser, and Thr;

⁴D is selected from the group consisting of Gly, Ala, Leu, Pro, Ile, and Val;

⁵E is selected from the group consisting of Ser, Pro and Thr; and

⁶F is selected from the group consisting of Ser, Thr or is absent, wherein ¹A is attached at the C-terminal amino acid sequence of the cellulase core and ⁶F is attached at the N-terminal amino acid sequence of the linker and/or domain (CBD).

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

¹Gly-²B-³Ile-⁴D-⁵Ser-⁶F

wherein

²B is Pro, Gln, or Glu;

⁴D is Gly or Pro;

⁵E is Ser; and

⁶F is Ser or is absent.

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

¹Val-²Gln-³Ile-⁴Pro-⁵Ser-⁶Ser.

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

¹Gly-²Glu-³Ile-⁴Gly-⁵Ser or ¹Gly-²Pro-³Ile-⁴Gly-⁵Ser.

6. 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.

7. The cellulase fusion protein of claim 6, 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.

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

9. The cellulase fusion protein of claim 8, 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.

10. The cellulase fusion protein of claim 9, wherein said cellulase is Melanocarpus albomyces 20 K cellulase, 50K cellulase, 50KB cellulase, or a derivative thereof.

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

12. The cellulase fusion protein of claim 6, wherein said acid cellulase is derived from Trichoderma sp. or Hypocrea.

13. The cellulase fusion protein of claim 6, wherein said neutral cellulase is the 20 K cellulase of Melanocarpus albomyces of SEQ ID NO:

2 or a derivative thereof, and the second amino acid sequence is the linker and/or CBD of Trichoderma reesei cellobiohydrolase I of SEQ ID NO;

4 or a derivative thereof.

14. The cellulase fusion protein of claim 1, wherein said first amino acid sequence, said second amino acid sequence, or both said first amino acid sequence and said second amino acid sequence are modified.

15. The cellulase fusion protein of claim 14, wherein in said second amino acid sequence, amino acid tyrosine at position 492 (position 31 of the CBD) and/or 493 (position 32 of the CBD) of the mature Trichoderma reesei CBHI is substituted with an aliphatic amino acid, and/or with an aromatic amino acid.

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

45, 46, 47, and 48.

17. The cellulase fusion protein of claim 14, wherein in said second amino acid sequence amino acids 434 to 444 or 434 to 460 of the mature Trichoderma reesei CBHI sequence are deleted.

18. The cellulase fusion protein of claim 17, wherein said second amino acid sequence is SEQ ID NO:

49 or 50.

19. The cellulase fusion protein of claim 14, wherein in said first amino acid sequence amino acid valine at position 208 of 20K cellulase sequence of Melanocarpus albomyces of SEQ ID NO:

2 has been deleted.

20. The cellulase fusion protein of claim 14, wherein in said first amino acid sequence amino acid alanine at position 207 of 20K cellulase sequence of Melanocarpus albomyces of SEQ ID NO:

2 has been deleted.

21. The cellulase fusion protein of claim 14, wherein in said first amino acid sequence amino acid phenylalanine at position 209 of 20 K cellulase sequence of Melanocarpus albomyces of SEQ ID NO:

2 has been substituted with Trp.

22. The cellulase fusion protein of claim 14, wherein in said first amino acid sequence amino acid contains an inserted proline after position 206 in 20 K cellulase sequence of Melanocarpus albomyces of SEQ ID NO:

2.

23. The cellulase fusion protein of claim 14, wherein in said first amino acid sequence amino acid is selected from the group consisting of SEQ ID NO:

37, 38, 39, and 40.

24. The cellulase fusion protein of claim 1, wherein said fusion protein is stable.

25. The cellulase fusion protein of claim 1, wherein a preparation containing said fusion protein has further been stabilized by heat treatment.

26. The cellulase fusion protein of claim 1, wherein said first cellulase naturally lacks a CBD.

27. The cellulase fusion protein of claim 1, wherein a CBD has been deleted from said first cellulase.

45. A process for the production of the cellulase fusion protein of claim 1 comprising the steps of culturing the host cell and recovering the protein from the culture medium.

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

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

48. The process of claim 47, wherein the fabric or garment is denim.

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

50. The process of claim 49, wherein the textile material is manufactured of natural cellulose containing fibers or manmade cellulose containing fibers or a mixture thereof.

51. The process of claim 49, wherein the textile material is a blend of synthetic fibers and cellulose containing fibers.

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

53. A method of treating cellulosic fiber containing textile material, wherein said method comprises contacting said textile material with the detergent composition of claim 52.

54. 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.

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

56. The cellulase fusion protein of claim 2, wherein

¹A is Gly or Val;

²B is Pro, Gln, or Glu;

³C is Ile;

⁴D is Gly or Pro;

⁵E is Ser; and

⁶F is Ser or is absent.

57. The cellulase fusion protein of claim 56, wherein

¹A is Gly.

58. The cellulase fusion protein of claim 9, wherein the natural cellulase is from a Melanocarpus sp.

59. The cellulase fusion protein of claim 9, wherein the natural cellulase is from Melanocarpus albomyces.

60. The cellulase fusion protein of claim 12, wherein said acid cellulase is derived from Trichoderma reesei.

61. The cellulase fusion protein of claim 15, wherein the aliphatic amino acid is alanine and the aromatic amino acid is tryptophan.

65. The process for biofinishing of claim 49, wherein said textile material is fabric, garment or yarn.

66. The detergent composition of claim 52, wherein said auxiliaries are selected from the group consisting of surface active agents, surfactants, bleaching agents and builders.

28. An expression vector comprising a first polynucleotide sequence encoding an optionally modified first amino acid sequence of a cellulase core derived from one species, and a second polynucleotide sequence encoding an optionally modified second amino acid sequence of a linker and/or cellulose binding domain (CBD) derived from another species, and a polynucleotide encoding a junction region connecting said first and second polynucleotide sequences, said polynucleotide sequences encoding the respective amino acid sequences as specifically defined above.

29. The expression vector of claim 28, wherein the first polynucleotide sequence encodes a neutral cellulase core and said second polynucleotide sequence encodes a linker and/or cellulose binding domain (CBD) of an acid cellulose.

30. The expression vector of claim 29, 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 or Chaetomium.

31. The expression vector of claim 30, wherein said cellulase is Melanocarpus albomyces 20 K cellulase, 50K cellulase, 50KB cellulase, or a derivative thereof.

32. The expression vector of claim 30, wherein said cellulase is Thermoascus aurantiacus CBHI.

33. The expression vector of claim 28, wherein said acid cellulase is derived from Trichoderma sp. or Hypocrea.

34. The expression vector of claim 28, wherein said neutral cellulase is the 20 K cellulase of Melanocarpus albomyces of SEQ ID NO:

1 or a derivative thereof, and the second amino acid sequence is the linker and/or CBD of Trichoderma reesei cellobiohydrolase I of SEQ ID NO;

3 or a derivative thereof.

35. The expression vector of claim 28, wherein said first amino acid sequence and/or said second amino acid sequence is modified.

36. The expression vector of claim 35, wherein said second polynucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:

45, 46, 47, and 48.

38. The expression vector of claim 35, wherein said first polynucleotide encodes an amino acid sequence selected from the group consisting of SEQ ID NO:

37, 38, 39, and 40.

39. The expression vector of claim 28, wherein said first polynucleotide encodes a cellulase which naturally lacks a CBD.

40. A host cell comprising the expression vector of claim 28.

41. The host cell of claim 40, which is of fungal origin.

42. The host cell of claim 41, which belongs to filamentous fungi.

43. The host cell of claim 41, which belongs to the genus Trichoderma or Aspergillus.

44. The host cell of claim 43, which is Trichoderma reesei.

62. The expression vector of claim 30, wherein said neutral cellulase is derived from a Melanocarpus sp.

63. The expression vector of claim 30, wherein said neutral cellulase is derived from Melanocarpus albomyces.

64. The expression vector of claim 33, wherein said acid cellulase is derived from Trichoderma reesei.

37. The expression vector of 35, wherein said second polynucleotide sequence encodes an amino acid sequence selected from SEQ ID NO:

49 or 50.