Variants of polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-9816082-B2
Application number	US-201113876788-A
Country	US
Kind code	B2
Filing date	Sep 29, 2011
Priority date	Sep 30, 2010
Publication date	Nov 14, 2017
Grant date	Nov 14, 2017

Abstract

The present invention relates to polypeptide having cellulolytic enhancing activity variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

First claim

What is claimed is:

1. A variant polypeptide of a parent GH61 polypeptide, comprising a substitution at one or more positions corresponding to positions 75, 77, 179, 181, and 183 of the full-length polypeptide of SEQ ID NO: 2, wherein the variant has cellulolytic enhancing activity, and wherein the parent GH61 polypeptide is selected from the group consisting of:
(a) a GH61 polypeptide having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2; and
(b) a GH61 polypeptide encoded by a polynucleotide having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

2. The variant of claim 1 , which comprises one or more substitutions selected from the group consisting of I75V, F77L, F179I, I181L, and I183V.

3. The variant of claim 1 , which comprises the substitutions I75V+F77L+F179I+I181L+I183V of the mature polypeptide of SEQ ID NO: 2.

4. An isolated polynucleotide encoding the variant of claim 1 .

5. An isolated recombinant host cell transformed with the polynucleotide of claim 4 .

6. A method of producing a variant, comprising:
(a) cultivating the recombinant host cell of claim 5 under conditions suitable for the expression of the variant; and
(b) recovering the variant.

7. A transgenic plant, plant part or plant cell transformed with the polynucleotide of claim 4 .

8. A method of producing the variant of claim 1 , comprising:
(a) transforming a plant or a plant cell with the polynucleotide encoding the variant;
(b) cultivating the transformed transgenic plant or a plant cell under conditions conducive for production of the variant; and
(c) recovering the variant from the cultivated and transformed transgenic plant or a plant cell.

9. A method for obtaining a variant, comprising introducing into a parent GH61 polypeptide a substitution at one or more positions corresponding to positions 75, 77, 179, 181, and 183 of SEQ ID NO: 2, wherein the variant has cellulolytic enhancing activity, and wherein the parent GH61 polypeptide is selected from the group consisting of:
(a) a GH61 polypeptide having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2; and
(b) a GH61 polypeptide encoded by a polynucleotide having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

10. A method for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition comprising the variant of claim 1 .

11. The method of claim 10 , further comprising recovering the degraded cellulosic material.

12. A method for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition comprising the variant of claim 1 ;
(b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.

13. A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition comprising the variant of claim 1 .

14. The method of claim 13 , wherein the fermenting of the cellulosic material produces a fermentation product.

15. The method of claim 14 , further comprising recovering the fermentation product from the fermentation.

16. A detergent composition comprising the variant of claim 1 and a surfactant.

17. A whole broth formulation or cell culture composition comprising the variant of claim 1 .

18. The variant of claim 1 , wherein the parent GH61 polypeptide has at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.

19. The variant of claim 1 , wherein the parent GH61 polypeptide comprises SEQ ID NO: 2 or the mature polypeptide thereof.

20. The variant of claim 1 , wherein the parent GH61 polypeptide consists of SEQ ID NO: 2 or the mature polypeptide thereof.

21. The variant of claim 1 , wherein the parent GH61 polypeptide is encoded by a polynucleotide having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

22. The variant of claim 1 , wherein the number of substitutions is 1, 2, 3, 4, or 5 substitutions.

23. The variant of claim 1 , which comprises a substitution at a position corresponding to position 75.

24. The variant of claim 23 , wherein the substitution is Val.

25. The variant of claim 1 , which comprises a substitution at a position corresponding to position 77.

26. The variant of claim 25 , wherein the substitution is Leu.

27. The variant of claim 1 , which comprises a substitution at a position corresponding to position 179.

28. The variant of claim 27 , wherein the substitution is Ile.

29. The variant of claim 1 , which comprises a substitution at a position corresponding to position 181.

30. The variant of claim 29 , wherein the substitution is Leu.

31. The variant of claim 1 , which comprises a substitution at a position corresponding to position 183.

32. The variant of claim 31 , wherein the substitution is Val.

33. The variant of claim 1 , which comprises a substitution at two positions corresponding to any of positions 75, 77, 179, 181, and 183.

34. The variant of claim 1 , which comprises a substitution at three positions corresponding to any of positions 75, 77, 179, 181, and 183.

35. The variant of claim 1 , which comprises a substitution at four positions corresponding to any of positions 75, 77, 179, 181, and 183.

36. The variant of claim 1 , which comprises a substitution at each position corresponding to positions 75, 77, 179, 181, and 183.

37. The variant of claim 1 , which has increased thermal activity, increased thermostability, or a combination of increased thermal activity and increased thermostability.

38. The method of claim 10 , wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

39. The method of claim 12 , wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

40. The method of claim 13 , wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a polypeptide having cellulolytic enhancing activity, a hemicellulase, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

41. The variant of claim 1 , wherein the parent GH61 polypeptide has at least 96% sequence identity to the mature polypeptide of SEQ ID NO: 2.

42. The variant of claim 1 , wherein the par

Assignees

• Sweeney Matt
• Wogulis Mark
• Novozymes Inc

Inventors

• Sweeney Matt
• Wogulis Mark

Classifications

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12N9/2437 Primary

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C12Y302/01004

  Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase · CPC title

• D21C5/005

  Treatment of cellulose-containing material with microorganisms or enzymes · CPC title

• C12N15/8202

  by biological means, e.g. cell mediated or natural vector · CPC title