Protease variants and compositions
View Patent ↗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 autoproteolytic stability in comparison to their wild type parent enzymes.
1. A modified subtilase comprising a substitution at position 134 and a substitution at position 190, wherein each position is numbered according to the amino acid sequence of the mature subtilisin BPN′ of SEQ ID NO: 36 and the modified subtilase has protease activity.
2. The modified subtilase of claim 1 , further comprising a substitution at one or more of positions 129, 130, 131, and 133.
3. The modified subtilase of claim 2 , comprising one or more substitutions selected from the group consisting of:
(a) a substitution at position 129 with F, I, L, M, or V; and
(b) a substitution at position 131 with F, I, L, M, or V.
4. The modified subtilase of claim 1 , further comprising a substitution at one or more of positions 159, 184, 185, 187, 170, and 171.
5. The modified subtilase of claim 4 , comprising one or mare of the following mutations:
(a) a substitution at position 159 with F, I, L, M, or V;
(b) a substitution at position 164 with F, I, L, M, or V;
(c) a substitution at position 167 with A, C, F, G, H, I, L, M, N, P, Q, S, T, V, or W;
(d) a substitution at position 170 with A, C, F, G, H, I, L, M, N, P, Q, S, T, V, W, or Y; and
(e) a substitution at position 171 with A, C, F, G, H, I, L, M, N, P, Q, S, T, V, or W.
6. The modified subtilase of claim 1 , further comprising a substitution at position 189.
7. The modified subtilase of claim 1 , comprising at least one further mutation at one or more other positions, wherein said further mutation is selected from the group consisting of a substitution, insertion or deletion.
8. The modified subtilase of claim 7 , wherein the one or more other positions are selected from the group consisting of: 27, 36, 57, 76, 97, 101, 104, 120, 123, 206, 218, 222, 224, 235 and 274.
9. The modified subtilase of claim 8 , wherein the subtilase is an I-S2 sub-group subtilase and the at least one further mutation is selected from the group consisting of K27R, *36D S57P, N76D, G97N, S101G, V104A, V104N, V104Y, H120D, N123S, Q206E, N218S, M222A, M222S, T224S, K235L, and T274A.
10. The modified subtilase of claim 9 , wherein the one at least one further mutation is selected from the group consisting of K27R+V104Y+N123S+T274A, N76D+V104A, V104N+S101G, and any other combination of K27R, N76D, S101G, V104A, V104N, V104Y, N123S, and T274A.
11. The modified subtilase of claim 1 , wherein the subtilase is a sub-group I-S1 subtilase.
12. The modified subtilase of claim 11 , wherein the subtilase is subtilisin I168, subtilisin BPN′, subtilisin DY, or subtilisin Carlsberg.
13. The modified subtilase of claim 1 , wherein the subtilase is a sub-group I-S2 subtilase.
14. The modified subtilase of claim 13 , wherein the subtilase is subtilisin 147, subtilisin 309, subtilisin PB92, or subtifisin YaB.
15. The modified subtilase of claim 13 , wherein the subtilase is thermitase.
16. A detergent composition comprising a modified subtilase of claim 1 and a surfactant.
17. The detergent composition of claim 16 , which additionally comprises an amylase, cellulase, cutinase, lipase, oxidoreductase, or another protease.
18. A DNA sequence encoding a modified subtilase of claim 1 .
19. A vector comprising a DNA sequence of claim 18 .
20. A microbial host cell transformed with a vector of claim 19 .
21. A method for producing a modified subtilase, comprising
(a) culturing a host of claim 20 under conditions conducive to the expression and secretion of the modified subtilase, and
(b) recovering the modified subtilase.