IP Library › Granted Patent US 8,008,056

Granted Patent B2

US 8,008,056 · App. 11/317,110 · Granted Aug 30, 2011

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Assignee: Danisco US Inc.

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Quick Facts

Patent No.: US 8,008,056
App. No.: 11/317,110
Granted: Aug 30, 2011
Kind: B2

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Abstract

Described herein are variants of H. jecorina CBH2, a Cel6A enzyme. The present invention provides novel cellobiohydrolases that have altered thermostability.

Claims (241)

1. An isolated variant of a CBH2 cellulase comprising SEQ ID NO:2 or SEQ ID NO:95, wherein the variant consists one or more substitutions as compared to SEQ ID NO:2 or SEQ ID NO:95, respectively, wherein said one or more substitutions are selected, in the context of SEQ ID NO:95, from the group consisting of: V94E, P98L, G118P, M120L, M134(G/L/V), T142V, L144(G/R/S), M145L, T148Y, T154A, L179A, Q204E, V206L, S210(L/R), I212V, T214(M/Y), L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A, T312S, S316P, V323(L/N/Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and A429T.

2. The variant of claim 1 which consists, in the context of SEQ ID NO:95, the substitution S316P and optionally one or more further substitutions selected from the group consisting of:

i. I212V/F411Y;

ii. M134G/L144G;

iii. M134L/L144R;

iv. M134L/L144S;

v. M134V/V206L/I212V/T312S/F411Y/S413Y;

vi. P98L/M134L/L144R/S210L/T214Y/V323Y/S413Y;

vii. P98L/M134L/L144R/S210R/T214Y/V323Y/S413Y;

viii. P98L/M134L/L144R/S413Y;

ix. P98L/M134L/L144R/V323Y/S413Y;

x. P98L/M134L/L144R/V206L/S210R/T214Y/S413Y;

xi. P98L/M134L/L144R/V206L/S210R/T214Y/V323Y/S413Y;

xii. P98L/M134V/I212V/S413Y;

xiii. P98L/M134V/I212V/T312S/S413Y;

xiv. P98L/M134V;

xv. P98L/M134V/S413Y;

xvi. P98L/M134V/V323Y/S413Y;

xvii. P98L/M134V/T154A/I212V/F411Y/S413Y;

xviii. P98L/M134V/T154A/I212V/S413Y;

xix. P98L/M134V/T154A/I212V/T312S/S413Y;

xx. P98L/M134V/T154A/V206L/I212V/F411Y/S413Y;

xxi. P98L/M134V/T154A/V206L;

xxii. P98L/M134V/V206L/I212V/F411Y;

xxiii. P98L/M134V/V206L/I212V/T312S/S413Y;

xxiv. P98L/M134V/V206L/S210R/T214Y/S413Y;

xxv. P98L/M134V/V206L/S210R/T214Y/V323Y/S413Y;

xxvi. P98L/M134V/V206L/S413Y;

xxvii. P98L/V206L/I212V/T312S/F411Y/S413Y;

xxviii. S413Y;

xxix. V323L;

xxx. V323Y;

xxxi. V206L/I212V;

xxxii. V206L/I212V/T312S;

xxxiii. V206L/I212V/T312S/F411Y/S413Y;

xxxiv. V206L/I212V/T312S/S413Y;

xxxv. V206L/S210L/T214M;

xxxvi. V206L/S210R;

xxxvii. V206L/S210R/T214Y; and

xxxviii. V206L.

3. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions I212V/S316P/F411Y.

4. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions M134G/L144G/S316P.

5. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions M134L/L144R/S316P.

6. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions M134L/L144S/S316P.

7. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions M134V/V206L/I212V/T312S/S316P/F411Y/S413Y.

8. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134L/L144R/S210L/T214Y/S316P/V323Y/S413Y.

9. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134L/L144R/S210R/T214Y/S316P/V323Y/S413Y.

10. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134L/L144R/S316P/S413Y.

11. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134L/L144R/S316P/V323Y/S413Y.

12. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134L/L144R/V206L/S210R/T214Y/S316P/S413Y.

13. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134L/L144R/V206L/S210R/T214Y/S316P/V323Y/S413Y.

14. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/I212V/S316P/S413Y.

15. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/I212V/T312S/S316P/S413Y.

16. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/S316P.

17. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/S316P/S413Y.

18. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/S316P/V323Y/S413Y.

19. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/T154A/I212V/S316P/F411Y/S413Y.

20. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/T154A/I212V/S316P/S413Y.

21. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/T154A/I212V/T312S/S316P/S413Y.

22. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/T154A/V206L/I212V/S316P/F411Y/S413Y.

23. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/T154A/V206L/S316P.

24. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/V206L/I212V/S316P/F411Y.

25. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/V206L/I212V/T312S/S316P/S413Y.

26. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/V206L/S210R/T214Y/S316P/S413Y.

27. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/V206L/S210R/T214Y/S316P/V323Y/S413Y.

28. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/M134V/V206L/S316P/S413Y.

29. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions P98L/V206L/I212V/T312S/S316P/F411Y/S413Y.

30. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions S316P/S413Y.

31. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions S316P/V323L.

32. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions S316P/V323Y.

33. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/I212V/S316P.

34. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/I212V/T312S/S316P.

35. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions 206L/I212V/T312S/S316P/F411Y/S413Y.

36. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/I212V/T312S/S316P/S413Y.

37. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/S210L/T214M/S316P.

38. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/S210R/S316P.

39. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/S210R/T214Y/S316P.

40. The variant CBH2 cellulase of claim 1 or claim 2 comprising the substitutions V206L/S316P.

41. A detergent composition, comprising a surfactant and a variant CBH2 cellulase according to claim 1 or claim 2 .

42. The detergent according to claim 41 , wherein said detergent is a laundry detergent.

43. The detergent according to claim 41 , wherein said detergent is a dish detergent.

44. A fungal cellulase composition enriched in a variant of a CBH2 cellulase comprising SEQ ID NO:2 or SEQ ID NO:95, wherein the variant consists one or more substitutions as compared to SEQ ID NO:2 or SEQ ID NO:95, respectively, wherein said one or more substitutions are selected, in the context of SEQ ID NO:95, from the group consisting of: V94E, P98L, G118P, M120L, M134(G/L/V), T142V, L144(G/R/S), M145L, T148Y, T154A, L179A, Q204E, V206L, S210(L/R), I212V, T214(M/Y), L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A, T312S, S316P, V323(L/N/Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and A429T.

45. The fungal cellulase composition of claim 44 wherein the variant consists, in the context of SEQ ID NO:95, the substitution S316P and optionally one or more further substitutions selected from the group consisting of:

i. I212V/F411Y;

ii. M134G/L144G;

iii. M134L/L144R;

iv. M134L/L144S;

v. M134V/V206L/I212V/T312S/F411Y/S413Y;

vi. P98L/M134L/L144R/S210L/T214Y/V323Y/S413Y;

vii. P98L/M134L/L144R/S210R/T214Y/V323Y/S413Y;

viii. P98L/M134L/L144R/S413Y;

ix. P98L/M134L/L144R/V323Y/S413Y;

x. P98L/M134L/L144R/V206L/S210R/T214Y/S413Y;

xi. P98L/M134L/L144R/V206L/S210R/T214Y/V323Y/S413Y;

xii. P98L/M134V/I212V/S413Y;

xiii. P98L/M134V/I212V/T312S/S413Y;

xiv. P98L/M134V;

xv. P98L/M134V/S413Y;

xvi. P98L/M134V/V323Y/S413Y;

xvii. P98L/M134V/T154A/I212V/F411Y/S413Y;

xviii. P98L/M134V/T154A/I212V/S413Y;

xix. P98L/M134V/T154A/I212V/T312S/S413Y;

xx. P98L/M134V/T154A/V206L/I212V/F411Y/S413Y;

xxi. P98L/M134V/T154A/V206L;

xxii. P98L/M134V/V206L/I212V/F411Y;

xxiii. P98L/M134V/V206L/I212V/T312S/S413Y;

xxiv. P98L/M134V/V206L/S210R/T214Y/S413Y;

xxv. P98L/M134V/V206L/S210R/T214Y/V323Y/S413Y;

xxvi. P98L/M134V/V206L/S413Y;

xxvii. P98L/V206L/I212V/T312S/F411Y/S413Y;

xxviii. S413Y;

xxix. V323L;

xxx. V323Y;

xxxi. V206L/I212V;

xxxii. V206L/I212V/T312S;

xxxiii. V206L/I212V/T312S/F411Y/S413Y;

xxxiv. V206L/I212V/T312S/S413Y;

xxxv. V206L/S210L/T214M;

xxxvi. V206L/S210R;

xxxvii. V206L/S210R/T214Y; and

xxxviii. V206L.

46. A culture medium comprising a variant of a CBH2 cellulase comprising SEQ ID NO:2 or SEQ ID NO:95, wherein the variant consists one or more substitutions as compared to SEQ ID NO:2 or SEQ ID NO:95, respectively, wherein said one or more substitutions are selected, in the context of SEQ ID NO:95, from the group consisting of: V94E, P98L, G118P, M120L, M134(G/L/V), T142V, L144(G/R/S), M145L, T148Y, T154A, L179A, Q204E, V206L, S210(L/R), I212V, T214(M/Y), L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A, T312S, S316P, V323(L/N/Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and A429T.

47. The culture medium of claim 46 , wherein the variant consists, in the context of SEQ ID NO:95, the substitution S316P and optionally one or more further substitutions selected from the group consisting of:

i. I212V/F411Y;

ii. M134G/L144G;

iii. M134L/L144R;

iv. M134L/L144S;

v. M134V/V206L/I212V/T312S/F411Y/S413Y;

vi. P98L/M134L/L144R/S210L/T214Y/V323Y/S413Y;

vii. P98L/M134L/L144R/S210R/T214Y/V323Y/S413Y;

viii. P98L/M134L/L144R/S413Y;

ix. P98L/M134L/L144R/V323Y/S413Y;

x. P98L/M134L/L144R/V206L/S210R/T214Y/S413Y;

xi. P98L/M134L/L144R/V206L/S210R/T214Y/V323Y/S413Y;

xii. P98L/M134V/I212V/S413Y;

xiii. P98L/M134V/I212V/T312S/S413Y;

xiv. P98L/M134V;

xv. P98L/M134V/S413Y;

xvi. P98L/M134V/V323Y/S413Y;

xvii. P98L/M134V/T154A/I212V/F411Y/S413Y;

xviii. P98L/M134V/T154A/I212V/S413Y;

xix. P98L/M134V/T154A/I212V/T312S/S413Y;

xx. P98L/M134V/T154A/V206L/I212V/F411Y/S413Y;

xxi. P98L/M134V/T154A/V206L;

xxii. P98L/M134V/V206L/I212V/F411Y;

xxiii. P98L/M134V/V206L/I212V/T312S/S413Y;

xxiv. P98L/M134V/V206L/S210R/T214Y/S413Y;

xxv. P98L/M134V/V206L/S210R/T214Y/V323Y/S413Y;

xxvi. P98L/M134V/V206L/S413Y;

xxvii. P98L/V206L/I212V/T312S/F411Y/S413Y;

xxviii. S413Y;

xxix. V323L;

xxx. V323Y;

xxxi. V206L/I212V;

xxxii. V206L/I212V/T312S;

xxxiii. V206L/I212V/T312S/F411Y/S413Y;

xxxiv. V206L/I212V/T312S/S413Y;

xxxv. V206L/S210L/T214M;

xxxvi. V206L/S210R;

xxxvii. V206L/S210R/T214Y; and

xxxviii. V206L.

48. A method of treating wood pulp comprising contacting said wood pulp with a variant of a CBH2 cellulase comprising SEQ ID NO:2 or SEQ ID NO:95, wherein the variant consists one or more substitutions as compared to SEQ ID NO:2 or SEQ ID NO:95, respectively, wherein said one or more substitutions are selected, in the context of SEQ ID NO:95, from the group consisting of: V94E, P98L, G118P, M120L, M134(G/L/V), T142V, L144(G/R/S), M145L, T148Y, T154A, L179A, Q204E, V206L, S210(L/R), I212V, T214(M/Y), L215I, G231N, T232V, V250I, Q276L, N285Q, S291G, G308A, T312S, S316P, V323(L/N/Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and A429T.

49. The method of claim 48 wherein the variant consists, in the context of SEQ ID NO:95, the substitution S316P and optionally one or more further substitutions selected from the group consisting of:

i. I212V/F411Y;

ii. M134G/L144G;

iii. M134L/L144R;

iv. M134L/L144S;

v. M134V/V206L/I212V/T312S/F411Y/S413Y;

vi. P98L/M134L/L144R/S210L/T214Y/V323Y/S413Y;

vii. P98L/M134L/L144R/S210R/T214Y/V323Y/S413Y;

viii. P98L/M134L/L144R/S413Y;

ix. P98L/M134L/L144R/V323Y/S413Y;

x. P98L/M134L/L144R/V206L/S210R/T214Y/S413Y;

xi. P98L/M134L/L144R/V206L/S210R/T214Y/V323Y/S413Y;

xii. P98L/M134V/I212V/S413Y;

xiii. P98L/M134V/I212V/T312S/S413Y;

xiv. P98L/M134V;

xv. P98L/M134V/S413Y;

xvi. P98L/M134V/V323Y/S413Y;

xvii. P98L/M134V/T154A/I212V/F411Y/S413Y;

xviii. P98L/M134V/T154A/I212V/S413Y;

xix. P98L/M134V/T154A/I212V/T312S/S413Y;

xx. P98L/M134V/T154A/V206L/I212V/F411Y/S413Y;

xxi. P98L/M134V/T154A/V206L;

xxii. P98L/M134V/V206L/I212V/F411Y;

xxiii. P98L/M134V/V206L/I212V/T312S/S413Y;

xxiv. P98L/M134V/V206L/S210R/T214Y/S413Y;

xxv. P98L/M134V/V206L/S210R/T214Y/V323Y/S413Y;

xxvi. P98L/M134V/V206L/S413Y;

xxvii. P98L/V206L/I212V/T312S/F411Y/S413Y;

xxviii. S413Y;

xxix. V323L;

xxx. V323Y;

xxxi. V206L/I212V;

xxxii. V206L/I212V/T312S;

xxxiii. V206L/I212V/T312S/F411Y/S413Y;

xxxiv. V206L/I212V/T312S/S413Y;

xxxv. V206L/S210L/T214M;

xxxvi. V206L/S210R;

xxxvii. V206L/S210R/T214Y; and

xxxviii. V206L.

50. A method of converting biomass to sugars comprising contacting said biomass with a variant of a CBH2 cellulase comprising SEQ ID NO:2 or SEQ ID NO:95, wherein the variant consists one or more substitutions as compared to SEQ ID NO:2 or SEQ ID NO:95, respectively, wherein said one or more substitutions are selected, in the context of SEQ ID NO:95, from the group consisting of: V94E, P98L, G118P, M120L, M134(G/L/V), T142V, L144(G/R/S), M145L, T148Y, T154A, L179A, Q204E, V206L, S210(L/R), I212V, T214(M/Y), L215I, G231N, T232V, V250I, Q276L, N285Q, 291 G, G308A, T312S, S316P, V323(L/N/Y), N325D, I333L, G334A, S343P, T349L, G360R, S380T, A381T, S386P, F411Y, S413Y, A416G, Q426E and A429T.

51. The method of claim 50 , wherein the variant consists, in the context of SEQ ID NO:95, the substitution S316P and optionally one or more further substitutions as compared to SEQ ID NO:2 or SEQ ID NO:95, respectively, wherein said one or more further substitutions are selected, in the context of SEQ ID NO:95, from the group consisting of:

i. I212V/F411Y;

ii. M134G/L144G;

iii. M134L/L144R;

iv. M134L/L144S;

v. M134V/V206L/I212V/T312S/F411Y/S413Y;

vi. P98L/M134L/L144R/S210L/T214Y/V323Y/S413Y;

vii. P98L/M134L/L144R/S210R/T214Y/V323Y/S413Y;

viii. P98L/M134L/L144R/S413Y;

ix. P98L/M134L/L144R/V323Y/S413Y;

x. P98L/M134L/L144R/V206L/S210R/T214Y/S413Y;

xi. P98L/M134L/L144R/V206L/S210R/T214Y/V323Y/S413Y;

xii. P98L/M134V/I212V/S413Y;

xiii. P98L/M134V/I212V/T312S/S413Y;

xiv. P98L/M134V;

xv. P98L/M134V/S413Y;

xvi. P98L/M134V/V323Y/S413Y;

xvii. P98L/M134V/T154A/I212V/F411Y/S413Y;

xviii. P98L/M134V/T154A/I212V/S413Y;

xix. P98L/M134V/T154A/I212V/T312S/S413Y;

xx. P98L/M134V/T154A/V206L/I212V/F411Y/S413Y;

xxi. P98L/M134V/T154A/V206L;

xxii. P98L/M134V/V206L/I212V/F411Y;

xxiii. P98L/M134V/V206L/I212V/T312S/S413Y;

xxiv. P98L/M134V/V206L/S210R/T214Y/S413Y;

xxv. P98L/M134V/V206L/S210R/T214Y/V323Y/S413Y;

xxvi. P98L/M134V/V206L/S413Y;

xxvii. P98L/V206L/I212V/T312S/F411Y/S413Y;

xxviii. S413Y;

xxix. V323L;

xxx. V323Y;

xxxi. V206L/I212V;

xxxii. V206L/I212V/T312S;

xxxiii. V206L/I212V/T312S/F411Y/S413Y;

xxxiv. V206L/I212V/T312S/S413Y;

xxxv. V206L/S210L/T214M;

xxxvi. V206L/S210R;

xxxvii. V206L/S210R/T214Y; and

xxxviii. V206L.

Assignments (3)

CHANGE OF NAME Recorded Jul 29, 2011

From: GENENCOR INTERNATIONAL, INC.

To: DANISCO US INC.

Reel/Frame 026672/0781 →

CORRECTIVE ASSIGNMENT TO CORRECT THE TO CORRECT THE SPELLING OF THE FIFTH ASSIGNOR'S NAME PREVIOUSLY RECORDED ON REEL 017634 FRAME 0706. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Jul 8, 2011

From: AEHLE, WOLFGANG; GOEDEGEBUUR, FRITS; DANKMEYER, LYDIA; NEEFE, PAULIEN; MITCHINSON, COLIN; KELEMEN, BRADLEY; CALDWELL, ROBERT; TEUNISSEN, PAULINE

To: GENENCOR INTERNATIONAL, INC.

Reel/Frame 026565/0657 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 16, 2006

From: AEHLE, WOLFGANG; GOEDEGEBUUR, FRITS; DANKMEYER, LYDIA; NEEFE, PAULLEN; MITHCHINSON, COLIN; KELEMEN, BRADLEY; CALDWELL, ROBERT; TEUNLSSEN, PAULINE

To: GENENCOR INTERNATIONAL, INC.

Reel/Frame 017634/0706 →

Continuity (3)

Provisional Application 60640398 · Dec 30, 2004

Provisional Application 60656863 · Feb 25, 2005

Related Publication 20060205042A1 · Sep 14, 2006