Protease variants and compositions

• US 6,682,924 B1
• Filed: 06/05/2000
• Issued: 01/27/2004
• Est. Priority Date: 05/05/1995
• Status: Expired due to Term

• Current Assignee: Novozymes A/S (Novozymes), Unilever PLC
• Original Assignee: Novozymes A/S (Novozymes), Unilever
• Inventors: Bauditz, Peter, Markvardsen, Peter, Sierkstra, Laurens Nicolaas, Klugkist, Jan, von der Osten, Claus
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US09/587,747
• Time in Patent Office: 1,331 Days
• Field of Search: 435/220, 435/221, 435/222, 435/69.1, 435/471
• US Class Current: 435/220
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C12N 9/54 bacteria being Bacillus

Abstract

The present invention relates to enzymes produced by mutating the genes for a number of subtilases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved stability and/or improved wash performance in any detergent in comparison to their wild type parent enzymes. The enzymes are well-suited for use in any detergent and for some in especially liquid or solid shaped detergent compositions.

Claims

1. A modified subtilisin 309 comprising one or both of the following substitutions:

(a) Y167A, Y167C, Y167G, Y167H, Y167I, Y167L, Y167M, Y167N, Y167P, Y167Q, Y167S, Y167T, or Y167W, and

(b) R170A, R170C, R170F, R170G, R170H, R170I, R170L, R170M, R170N, R170P, R170S, R170T, R170V, or R170W, wherein each position corresponds to a position of the amino acid sequence of subtilisin BPN′

.

2. The modified subtilisin 309 of claim 1, which comprises Y167A.

3. The modified subtilisin 309 of claim 1, which comprises Y167C.

4. The modified subtilisin 309 of claim 1, which comprises Y167G.

5. The modified subtilisin 309 of claim 1, which comprises Y167H.

6. The modified subtilisin 309 of claim 1, which comprises Y167I.

7. The modified subtilisin 309 of claim 1, which comprises Y167L.

8. The modified subtilisin 309 of claim 1, which comprises Y167M.

9. The modified subtilisin 309 of claim 1, which comprises Y167N.

10. The modified subtilisin 309 of claim 1, which comprises Y167P.

11. The modified subtilisin 309 of claim 1, which comprises Y167Q.

12. The modified subtilisin 309 of claim 1, which comprises Y167S.

13. The modified subtilisin 309 of claim 1, which comprises Y167T.

14. The modified subtilisin 309 of claim 1, which comprises Y167V.

15. The modified subtilisin 309 of claim 1, which comprises Y167W.

16. The modified subtilisin 309 of claim 1, which comprises R170A.

17. The modified subtilisin 309 of claim 1, which comprises R170C.

18. The modified subtilisin 309 of claim 1, which comprises R170F.

19. The modified subtilisin 309 of claim 1, which comprises R170G.

20. The modified subtilisin 309 of claim 1, which comprises R170H.

21. The modified subtilisin 309 of claim 1, which comprises R170I.

22. The modified subtilisin 309 of claim 1, which comprises R170L.

23. The modified subtilisin 309 of claim 1, which comprises R170M.

24. The modified subtilisin 309 of claim 1, which comprises R170N.

25. The modified subtilisin 309 of claim 1, which comprises R170P.

26. The modified subtilisin 309 of claim 1, which comprises R170Q.

27. The modified subtilisin 309 of claim 1, which comprises R170S.

28. The modified subtilisin 309 of claim 1, which comprises R170T.

29. The modified subtilisin 309 of claim 1, further comprising at least one further mutation at one or more of positions:

27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274.

30. The modified subtilisin 309 of claim 31, wherein at least one further mutation is selected from the group consisting of K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.

31. A detergent composition comprising a modified subtilisin 309 of claim 1 and a surfactant.

32. The detergent composition of claim 32, wherein the composition is in solid powdered form.

33. The detergent composition of claim 32, wherein the composition is in liquid form.

34. An isolated nucleic acid encoding a modified subtilisin 309 of claim 1.

35. A vector comprising a nucleic acid of claim 34.

36. A microbial host cell comprising a vector of claim 35.

37. A method for producing a modified subtilisin 309, which comprises

(a) culturing a microbial host cell of claim 36 under conditions conducive to the expression and secretion of the modified subtilisin 309, and

(b) recovering the modified subtilisin 309.

38. A modified Bacillus protease PB92 comprising one or both of the following substitutions:

(a) Y167A, Y167C, Y167G, Y167H, Y1671, Y167L, Y167M, Y167N, Y167P, Y167Q, Y167S, Y167T, or Y167W, and

(b) R170A, R170C, R170F, R170G, R170H, R170L, R170N, R170P, R170S, R170T, R1 70W, or R170Y, wherein each position corresponds to a position of the amino acid sequence of subtilisin BPN′

.

39. The modified Bacillus protease PB92 of claim 38, further comprising at least one further mutation at one or more of positions:

27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274.

40. The modified Bacillus protease PB92 of claim 39, wherein at least one further mutation is selected from the group consisting of K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.

41. A detergent composition comprising a modified Bacillus protease PB92 of claim 38 and a surfactant.

42. The detergent composition of claim 41, wherein the composition is in solid powdered form.

43. The detergent composition of claim 41, wherein the composition is in liquid form.

44. An isolated nucleic acid encoding a modified Bacillus protease PB92 of claim 38.

45. A vector comprising a nucleic acid of claim 44.

46. A microbial host cell comprising a vector of claim 45.

47. A method for producing a modified Bacillus protease PB92, which comprises

(a) culturing a microbial host cell of claim 46 under conditions conducive to the expression and secretion of the modified Bacillus protease PB92, and

(b) recovering the modified Bacillus protease PB92.

48. A modified subtilisin 147 comprising one or both of the following substitutions:

(a) Y167A, Y167C, Y167G, Y167H, Y167I, Y167L, Y167M, Y167N, Y167P, Y167Q, Y167S, Y167T, or Y167W, and

(b) R170A, R170C, R170F, R170G, R170H, R170I R170L, R170M, R170N, R170P, R170S, R170T, R170V, R170W, or R170Y, wherein each position corresponds to a position of the amino acid sequence of subtilisin BPN′

.

49. The modified subtilisin 147 of claim 48, further comprising at least one further mutation at one or more of positions:

27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274.

50. The modified subtilisin 147 of claim 49, wherein at least one further mutation is selected from the group consisting of K27R, *36D, S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, A194P, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.

51. A detergent composition comprising a modified subtilisin 147 of claim 48 and a surfactant.

52. The detergent composition of claim 51, wherein the composition is in solid powdered form.

53. The detergent composition of claim 51, wherein the composition is in liquid form.

54. An isolated nucleic acid encoding a modified subtilisin 147 of claim 48.

55. A vector comprising a nucleic acid of claim 54.

56. A microbial host cell comprising a vector of claim 55.

57. A method for producing a modified subtilisin 147, which comprises

(a) culturing a microbial host cell of claim 56 under conditions conducive to the expression and secretion of the modified subtilisin 147, and

(b) recovering the modified subtilisin 147.

58. A modified subtilsin 309, comprising both of the following substitutions:

(a) Y167A, Y167C, Y167G, Y167H, Y167I, Y167L, Y167M, Y167N, Y167P, Y167Q, Y167S, Y167T, Y167V, or Y167W, and

(b) R170A, R170C, R170F, R170 G, R170H, R170I, R170L, R170M, R170N, R170P, R170Q, R170S, R170T, R170V, or R170W,

wherein each position corresponds to a position of the amino acid sequence of subtilsin BPN′

.

59. A modified Bacillus protease PB92 comprising both of the following substitutions:

(a) Y167A, Y167C, Y167G, Y167H, Y167I, Y167L, Y167M, Y167N, Y167P, Y167Q, Y167S, Y167T, Y167V, or Y167W, and

(b) R170A, R170C, R170F, R170 G, R170H, R170I, R170L, R170M, R170N, R170P, R170Q, R170S, R170T, R170V, or R170W,

wherein each position corresponds to a position of the amino acid sequence of subtilsin BPN′

.

60. A modified subtilsin 147, comprising both of the following substitutions:

(a) Y167A, Y167C, Y167G, Y167H, Y167I, Y167L, Y167M, Y167N, Y167P, Y167Q, Y167S, Y167T, Y167V, or Y167W, and

(b) R170A, R170C, R170F, R170 G, R170H, R170I, R170L, R170M, R170N, R170P, R170Q, R170S, R170T, R170V, or R170W,

wherein each position corresponds to a position of the amino acid sequence of subtilsin BPN′

.