IP Library › Granted Patent US 8,637,294
Granted Patent B2
US 8,637,294 · App. 11/728,219 · Granted Jan 28, 2014

Variant

Inventors: Anthony Day (San Francisco, CA); Frits Goedegebuur (Vlaardingen, NL); Peter Gualfetti (San Francisco, CA); Colin Mitchinson (Half Moon Bay, CA); Paulien Neefe (Zoetermeer, NL); Mats Sandgren (Uppsala, SE); Andrew Shaw (San Francisco, CA); Jerry Stahlberg (Uppsala, SE)
Assignee: Danisco US Inc.
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Quick Facts
Patent No.
US 8,637,294
App. No.
11/728,219
Granted
Jan 28, 2014
Kind
B2
Abstract

Described herein are variants of H. jecorina CBH I, a Cel7 enzyme. The present invention provides novel cellobiohydrolases that have improved thermostability and reversibility.

Claims (88)

1. A nucleic acid encoding a Hypocrea jecorina CBH I variant consisting essentially of an amino acid sequence having at least 96.4% sequence identity to the amino acid sequence of SEQ ID NO:2, and comprising one or more alterations as compared to SEQ ID NO:2, wherein said one or more alterations is selected from the group consisting of:

i. A112E/T226A;

ii. S196T/S411F;

iii. E295K/S398T;

iv. T246C/Y371C;

v. V403D/T462I;

vi. T41I plus deletion at T445;

vii. A68T/G440R/P491L;

viii. G22D/S278P/T296P;

ix. T246A/R251A/Y252A;

x. T380G/Y381D/R394A;

xi. Y252Q/D259W/S342Y;

xii. S113T/T255P/K286M;

xiii. P227L/E325K/Q487L;

xiv. P227T/T484S/F352L;

xv. Q17L/E193V/M213I/F352L;

xvi. S8P/N49S/A68T/S113N;

xvii. S8P/N49S/A68T/S113N/P227L;

xviii. T41I/A112E/P227L/S278P/T296P;

xix. S8P/N49S/A68T/A112E/T226A;

xx. S8P/N49S/A68T/A112E/P227L;

xxi. S8P/T41I/N49S/A68T/A112E/P227L;

xxii. G22D/N49S/A68T/P227L/S278P/T296P;

xxiii. S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P;

xxiv. G22D/N49S/A68T/N103I/S113N/P227L/S278P/T296P;

xxv. G22D/N49S/A68T/N103I/A112E/P227L/S278P/T296P;

xxvi. G22D/N49S/N64D/A68T/N103I/S113N/S278P/T296P;

xxvii. S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P;

xxviii. S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/S278P/T296P/N301R;

xxix. S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P/N301R;

xxx. S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/T296P/N301R;

xxxi. S8P/T41I/N49S/S57N/A68T/S113N/P227L/D249K/S278P/T296P/N301R;

xxxii. S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/N301R;

xxxiii. S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I;

xxxiv. S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I;

xxxv. S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F;

xxxvi. S 8P/G22D/T41I/N49S/A68T/S 92T/S113N/P227L/D249K/S411F;

xxxvii. S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/S411F;

xxxviii. S8P/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/V403D/S411F/T462I; and

xxxix. S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/V403D/S411F/T462I.

2. A vector comprising a nucleic acid of claim 1 .

3. A host cell transformed with the vector of claim 2 .

4. A method of producing a CBH I variant comprising the steps of:

(a) culturing the host cell according to claim 3 in a suitable culture medium under suitable conditions to produce a CBH I variant; and

(b) obtaining said produced CBH I variant.

5. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of A112E/T226A.

6. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of S196T/S411F.

7. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of E295K/S398T.

8. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of T246C/Y371C.

9. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of V403D/T462I.

10. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of T41I plus deletion at T445.

11. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of A68T/G440R/P491L.

12. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of G22D/S278P/T296P.

13. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of T246A/R251A/Y252A.

14. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of T380G/Y381D/R394A.

15. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of Y252Q/D259W/S342Y.

16. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of S113T/T255P/K286M.

17. The nucleic acid of claim 1 wherein said one or more alterations comprises P227L/E325K/Q487L.

18. The nucleic acid of claim 1 wherein said one or more alterations comprises P227T/T484S/F352L.

19. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of Q17L/E193V/M213I/F352L.

20. The nucleic acid of claim 1 wherein said one or more alterations comprises consists essentially of S8P/N49S/A68T/S113N.

21. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/N49S/A68T/S113N/P227L.

22. The nucleic acid of claim 1 wherein said one or more alterations comprises T41I/A112E/P227L/S278P/T296P.

23. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of S8P/N49S/A68T/A112E/T226A.

24. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/N49S/A68T/A112E/P227L.

25. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/T411/N49S/A68T/A112E/P227L.

26. The nucleic acid of claim 1 wherein said one or more alterations comprises G22D/N49S/A68T/P227L/S278P/T296P.

27. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T411/N49S/A68T/N103I/S113N/P227L/S278P/T296P.

28. The nucleic acid of claim 1 wherein said one or more alterations comprises G22D/N49S/A68T/N103I/S113N/P227L/S278P/T296P.

29. The nucleic acid of claim 1 wherein said one or more alterations comprises G22D/N49S/A68T/N103I/A112E/P227L/S278P/T296P.

30. The nucleic acid of claim 4 wherein said one or more alterations consists essentially of G22D/N49S/N64D/A68T/N103I/S113N/S278P/T296P.

31. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T411/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P.

32. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T411/N49S/A68T/N1031/S113N/P227L/S278P/T296P/N301R.

33. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T41I/N49S/A68T/N103I/S113N/P227L/D249K/S278P/T296P/N301R.

34. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/T296P/N301R.

35. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/T41I/N49S/S57N/A68T/S113N/P227L/D249K/S278P/T296P/N301R.

36. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T41I/N49S/A68T/S113N/P227L/D249K/S278P/N301R.

37. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I.

38. The nucleic acid of claim 1 wherein said one or more alterations consists essentially of comprises S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/V403D/T462I.

39. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F.

40. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T41I/N49S/A68T/S92T/S113N/P227L/D249K/S411F.

41. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/S411F.

42. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/V403D/S411F/T462I.

43. The nucleic acid of claim 1 wherein said one or more alterations comprises S8P/G22D/T41I/N49S/A68T/S92T/S113N/S196T/P227L/D249K/T255P/S278P/T296P/N301R/E325K/V403D/S411F/T462I.

44. A recombinant filamentous fungal cell engineered to be capable of expressing a CBH I variant encoded by the nucleic acid of claim 1 .

45. A method of producing a CBH I variant comprising the steps of:

(a) culturing the recombinant filamentous fungal cell according to claim 44 in a suitable culture medium under suitable conditions to express a CBH I variant; and

(b) obtaining said CBH I variant.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 16, 2011
From: DAY, ANTHONY; GOEDEGEBUUR, FRITS; GUALFETTI, PETER; MITCHINSON, COLIN; NEEFE, PAULIEN; SANDGREN, MATS; SHAW, ANDREW; STAHLBERG, JERRY
To: GENENCOR INTERNATIONAL, INC.
Reel/Frame 025966/0538 →
CHANGE OF NAME Recorded Mar 16, 2011
From: GENENCOR INTERNATIONAL, INC.
To: DANISCO US INC.
Reel/Frame 025967/0380 →
Continuity (6)
Division 10641678 · Aug 15, 2003
Provisional Application 60404063 · Aug 16, 2002
Provisional Application 60458853 · Mar 27, 2003
Provisional Application 60456368 · Mar 21, 2003
Provisional Application 60458696 · Mar 27, 2003
Related Publication 20070173431A1 · Jul 26, 2007