The present invention relates to a method of designing laccase mutants with increased oxidation potential and/or changed pH optimum and/or altered mediator pathway and/or altered O 2 /OH − -pathway, which method is based on the hitherto unknown three dimensional structure of laccases, wherein the parent laccase is a Myceliophthora thermophila, Polyporus pinsitus , or Coprinus cinereus laccase.
1. An isolated variant of a laccase, wherein the variant has laccase activity and has the amino acid sequence of SEQ ID NO: 10 except for one or more of the following mutations:
(a) H93E;
(b) H95E;
(c) D106A, D106F, D106I, D106L, D106P, D106V, D106W, or D106Y;
(d) A108F, A108I, A108L, A108P, A108W, or A108Y;
(e) N109A, N109D, N109F, N109I, N109L, N109P, N109Q, N109V, N109W, or N109Y;
(f) T428A, T428F, T428I, T428L, T428P, T428V, T428W, or T428Y;
(g) L500F, L500I, L500W, or L500Y;
(h) A506E;
(i) S510A, S510F, S510I, S510L, S510P, S510V, S510W, or S510Y;
(j) G511F, G511I, G511L, G511P, G511V, G511W, or G511Y; or
(k) G514A, G514F, G514I, G514L, G514P, G514V, G514W, or G514Y.
2. The variant of claim 1 , which comprises H93E.
3. The variant of claim 1 , which comprises H95E.
4. The variant of claim 1 , which comprises D106A, D106F, D106I, D106L, D106P, D106V, D106W, or D106Y.
5. The variant of claim 1 , which comprises A108F, A108I, A108L, A108P, A108W, or A108Y.
6. The variant of claim 1 , which comprises N109A, N109D, N109F, N109I, N109L, N109P, N109Q, N109V, N109W, or N109Y.
7. The variant of claim 1 , which comprises T428A, T428F, T428I, T428L, T428P, T428V, T428W, or T428Y.
8. The variant of claim 1 , which comprises L500F, L500I, L500W, or L500Y.
9. The variant of claim 1 , which comprises A506E.
10. The variant of claim 1 , which comprises S510A, S510F, S510I, S510L, S510P, S510V, S510W, or S510Y.
11. The variant of claim 1 , which comprises G511F, G511I, G511L, G511P, G511V, G511W, or G511Y.
12. The variant of claim 1 , which comprises G514A, G514F, G514I, G514L, G514P, G514V, G514W, or G514Y.
13. A detergent composition comprising the variant of claim 1 and a surfactant.
14. An isolated variant of a laccase, wherein the variant has laccase activity and has the amino acid sequence of SEQ ID NO: 10 except for a substitution at one or more of the following positions:
185, 187, 189, 191, 193, 234, 235, 236, 269, 293, and 294.
15. The variant of claim 14 , which comprises a substitution at position 185.
16. The variant of claim 14 , which comprises a substitution at position 187.
17. The variant of claim 14 , which comprises a substitution at position 189.
18. The variant of claim 14 , which comprises a substitution at position 191.
19. The variant of claim 14 , which comprises a substitution at position 193.
20. The variant of claim 14 , which comprises a substitution at position 234.
21. The variant of claim 14 , which comprises a substitution at position 235.
22. The variant of claim 14 , which comprises a substitution at position 236.
23. The variant of claim 14 , which comprises a substitution at position 269.
24. The variant of claim 14 , which comprises a substitution at position 293.
25. The variant of claim 14 , which comprises a substitution at position 294.
26. A detergent composition comprising the variant of claim 14 and a surfactant.
27. An isolated variant of a laccase, wherein the variant has laccase activity and has the amino acid sequence of SEQ ID NO: 10 except for a substitution at one or more of the following positions:
185, 187, 189, 191, 193, 234, 235, 236, 269, 293, and 294; and
a substitution at one or more of the following positions:
186, 188, 190, 192, and 194.
28. The variant of claim 27 , which comprises a substitution at position 186.
29. The variant of claim 27 , which comprises a substitution at position 188.
30. The variant of claim 27 , which comprises a substitution at position 190.
31. The variant of claim 27 , which comprises a substitution at position 192.
32. The variant of claim 27 , which comprises a substitution at position 194.
33. A detergent composition comprising the variant of claim 27 and a surfactant.