Protease variants and polynucleotides encoding same

Abstract

The present invention relates to protease variants and methods for obtaining protease variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Claims

1. A protease variant comprising the alteration S27K, wherein the position number corresponds to SEQ ID NO: 3, and wherein the protease variant has at least 90% sequence indentity to SEQ ID NO: 3.

2. The protease variant of claim 1, which has improved wash performance compared to the protease with SEQ ID NO: 3.

3. The protease variant of claim 1, wherein the variant is selected from the group consisting of: a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizes under medium or high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i) or (ii); c) a polypeptide encoded by a polynucleotide having at least 90% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2; and d) a protease variant having at least 90% identity to SEQ ID NO 3, and comprising an alteration selected from the group consisting of: V2H, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, I137E, S173P, S175P, F180Y, T297P; A1*, V2*, P3*, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; N16K, D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, I137E, S173P, S175P, F180Y, T297P; D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, I137E, S173P, S175P, F180Y, T297P; Q18K, S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, S175P; S27K, I137E, S173P, S175P, F180Y, T297P; S27K, S173P, S175P, Q198E; S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, S144R, S173P, S175P, F180Y, Q198E, T297P; S27K, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, I137E, S144R, S173P, S175P, F180Y, T297P; S27K, E127Q, I137E, S173P, S175P, F180Y, T297P; S27K, I137E, S173P, G174R, S175P, F180Y, T297P; S27K, Q97K, I137E, S173P, S175P, F180Y, T297P; D17N, S27K, I137E, S173P, S175P, F180Y, T297P; S27K, D126G, S173P, S175P, F180Y, T297P; S27K, Y39R, S173P, S175P, F180Y, Q198E, T297P; S27K, Y39R, I137E, S173P, S175P, F180Y, T297P; S27K, Y39R, I137E, S144R, S173P, S175P, F180Y, T297P; S27K, Y39R, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S41K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P; E50T, S173P, S175P, F180Y; E50L, S173P, S175P, F180Y; G28N, Q57K, S86K, N87R, Q89R, S173P, S175P; S27K, S65K, S173P, S175P, F180Y, T297P; S27K, S65K, S173P, S175P, F180Y, T297P; S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P; S27K, Q97K, I137E, S173P, S175P, F180Y, T297P; S27K, N59L, T67V, Q97K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P; S27K, S111R, A118R, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, I137Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S144R, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162S, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162E, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162G, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162F, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162T, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162Y, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, I137E, S173P, G174R, S175P, F180Y, T297P; S27K, E127Q, S173P, S175P, F180Y, E194S, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, E194K, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, E194Q, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, N195H, V196N, Q197*, Q198*, N199*, G200*, T201*, T297P; S27K, S173P, S175P, F180Y, Q197K, T297P; S27K, S173P, S175P, F180Y, E194R, Q197K, Q198E, T297P; S27K, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, S173P, G174K, S175P, F180Y, L184F, Q197K, Q198E, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q197R, Q198E, T297P; S27K, Q97K, E127Q, S173P, S175P, F180Y, Q198E, D225N, I247M, T297P; T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, P3*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; S27K, S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, I137E, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, Q198E, I293R, T297P; S27K, I137E, S173P, S175P, F180Y, I293R, T297P; S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q197K, Q198E, S274V, T297P; S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, D147S, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S111R, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P; S27K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; and S4K, S27K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P.

4. A detergent composition comprising a protease variant according to claim 1 and one or more detergent components.

5. The detergent composition according to claim 4, further comprising one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof.

6. The detergent composition according to claim 4 in the form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

7. The variant of claim 1, wherein the variant has at least 91% sequence identity to SEQ ID NO: 3.

8. The variant of claim 1, wherein the variant has at least 92% sequence identity to SEQ ID NO: 3.

9. The variant of claim 1, wherein the variant has at least 93% sequence identity to SEQ ID NO: 3.

10. The variant of claim 1, wherein the variant has at least 94% sequence identity to SEQ ID NO: 3.

11. The variant of claim 1, wherein the variant has at least 95% sequence identity to SEQ ID NO: 3.

12. The variant of claim 1, wherein the variant further comprises an alteration selected from the group consisting of V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, 1220K, 1220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, 1247K, S248K, 1264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, 1293K, T297K, D299R and K311R.

13. The protease variant of claim 12, wherein the total number of alterations compared to SEQ ID NO: 3 is 1-20.

14. The variant of claim 12, wherein the variant further comprises a set of alterations selected from the group consisting of: S27K, N109K, S173P, S175P, F180Y, Q198E, T297P; S27K, N109K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N87K, S173P, S175P, F180Y, Q198E, T297P; S27K, N87K, I137E, S173P, S175P, F180Y, T297P; S27K, N87K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P; S27K, N59L, T67V, N87K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N59L, T67V, N87K, G107N, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, Q70N, N87K, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, N199R, T297P; S27K, N87K, I121V, E127Q, S171N, S173P, S175P, F180Y, T297P; S4K, D17N, S27K, N59L, N87K, Y156R, S173P, S175P, F180Y, Q198E, T297P; S4K, D17N, S27K, N59L, N87K, Y156R, S173P, S175P, F180Y, Q198E, T297P; S27K, N109K, S111D, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, N109K, S111E, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, Y39R, N87K, I137E, S173P, S175P, F180Y, T297P; S4K, S27K, N87K, E127Q, G132H, S173P, S175P, F180Y, Q198E, T297P; S27K, N59L, T67V, N87K, S114V, I121V, I150L, S171N, S173P, G174R, S175P, F180Y, T297P; S27K, N87K, I121V, S171N, S173P, S175P, F180Y, S270K, T297P; S27K, N87K, G132H, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, N87K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P; S27K, N87K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N87K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, N87K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P; S27K, N87K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S4K, S27K, N87K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P; S27K, K146E, D147S, S171N, S173P, G174R, S175P, F180Y, G182A, Q198E, N199K, E223K, T297P; S27K, S173P, S175P, F180Y, E194*, Q197K, Q198E, N199K, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, S173P, S175P, F180Y, Q197K, Q198E, N199K, T297P; S27K, Q70N, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S114V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, Q70N, G107N, I121V, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, Q70A, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, Q70V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S171R, S173P, G174E, S175P, F180Y, Q198E, N199K, T297P; S27K, S171K, S173P, G174E, S175P, F180Y, Q198E, N199K, T297P; S27K, S171E, S173P, G174K, S175P, F180Y, Q198E, N199K, T297P; S27K, D147N, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P; S27K, G142E, D147N, I150S, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, G142E, I150S, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P; S27K, N109S, *109aE, S111R, S171E, S173P, G174K, S175P, F180Y, G182A, L184FQ198E, N199K, T297P; S27K, N109S, *109aE, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, D147N, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, N109K, S111E, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, S111E, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, S111D, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, S111E, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, S114V, I121V, S129K, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P; S27K, S114V, I121V, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P; and S27K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P.

15. The protease variant of claim 12, wherein the variant is selected from the group consisting of: a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizes under medium or high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i) or (ii); c) a polypeptide encoded by a polynucleotide having at least 90% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2; and d) a protease variant having at least 90% identity to SEQ ID NO 3, and comprising an alteration selected from the group consisting of: V2H, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, I137E, S173P, S175P, F180Y, T297P; A1*, V2*, P3*, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; N16K, D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, I137E, S173P, S175P, F180Y, T297P; D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, I137E, S173P, S175P, F180Y, T297P; Q18K, S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, S175P; S27K, I137E, S173P, S175P, F180Y, T297P; S27K, S173P, S175P, Q198E; S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, S144R, S173P, S175P, F180Y, Q198E, T297P; S27K, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E500, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, I137E, S144R, S173P, S175P, F180Y, T297P; S27K, E127Q, I137E, S173P, S175P, F180Y, T297P; S27K, I137E, S173P, G174R, S175P, F180Y, T297P; S27K, Q97K, I137E, S173P, S175P, F180Y, T297P; D17N, S27K, I137E, S173P, S175P, F180Y, T297P; S27K, D126G, S173P, S175P, F180Y, T297P; S27K, Y39R, S173P, S175P, F180Y, Q198E, T297P; S27K, Y39R, I137E, S173P, S175P, F180Y, T297P; S27K, Y39R, I137E, S144R, S173P, S175P, F180Y, T297P; S27K, Y39R, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S41K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P; S27K, S65K, S173P, S175P, F180Y, T297P; S27K, S65K, S173P, S175P, F180Y, T297P; S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P; S27K, Q97K, I137E, S173P, S175P, F180Y, T297P; S27K, N59L, T67V, Q97K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P; S27K, S111R, A118R, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, I137Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S144R, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162S, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162E, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162G, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162F, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162T, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162Y, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, I137E, S173P, G174R, S175P, F180Y, T297P; S27K, E127Q, S173P, S175P, F180Y, E194S, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, E194K, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, E194Q, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, N195H, V196N, Q197*, Q198*, N199*, G200*, T201*, T297P; S27K, S173P, S175P, F180Y, Q197K, T297P; S27K, S173P, S175P, F180Y, E194R, Q197K, Q198E, T297P; S27K, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, S173P, G174K, S175P, F180Y, L184F, Q197K, Q198E, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q197R, Q198E, T297P; S27K, Q97K, E127Q, S173P, S175P, F180Y, Q198E, D225N, I247M, T297P; T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, P3*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, N268K, T297P; S27K, S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, I137E, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, Q198E, I293R, T297P; S27K, I137E, S173P, S175P, F180Y, I293R, T297P; S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q197K, Q198E, S274V, T297P; S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, D147S, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S111R, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P; S27K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; and S4K, S27K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P.

16. A method for obtaining a protease variant, comprising introducing into a parent protease the alteration S27K, wherein the position number corresponds to SEQ ID NO 3, wherein the protease variant has an amino acid sequence which is at least 90% identical to SEQ ID NO: 3, and recovering the variant.

17. The method according to claim 16 wherein the protease variant has at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 3.

18. The method according to claim 16, wherein the parent protease selected from the group consisting of: a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizes under medium or high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i) or (ii); c) a polypeptide encoded by a polynucleotide having at least 90% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention provides novel protease variants obtained from Bacillus sp., in particular, Bacillus sp. TY-145. The protease variants of the invention comprise at least 60% sequence identity to a polypeptide with SEQ ID NO 3 and comprise a substitution of at least one amino acid position compared to the protease with SEQ ID NO: 3, the at least one amino acid position selected from the group consisting of positions 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297, 299 and 311.

(2) One embodiment of the invention relates to protease variants having at least 60% sequence identity to SEQ ID NO 3, having proteolytic activity and comprising one or more alterations selected from the group consisting of V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 3. In a preferred embodiment the protease variant comprises one or more alterations selected from the group consisting of V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 3 and wherein the variant has at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, e.g. at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6, but less than 100%, sequence identity to SEQ ID NO: 3.

(3) The isoelectric point, pI or pH(I), is the pH value where the enzyme molecule is neutral, i.e. the sum of electrostatic charges (net electrostatic charge, =NEC, or net charge) of the molecule is equal to zero. Positive or negative nature of the individual electrostatic charges must be taken into account. The isoelectric point may be calculated by using equilibrium considerations using pKa values for the various charged residues in the enzyme in question and then by iteration find the pH value where the NEC of the enzyme molecule is equal to zero.

(4) The net charge of the molecule is affected by the pH of its surrounding environment and can become larger or smaller due to the gain or loss, respectively, of protons (H.sup.+).

(5) The pK.sub.a values for the charged residues are dependent on their environment and are consequently subject to variation. However, specific approximate pK.sub.a values may be allocated to the charged residues independently of the actual value. The pI of an enzyme molecule may also be determined experimentally by isoelectric focusing or by titrating a solution containing the enzyme. Various pK.sub.a values for the charged residues may be determined experimentally by titration.

(6) It has previously been shown in WO91/000345 (Novozymes A/S) that changes in the pI of a protease by making specific substitutions to produce a protease variants having altered overall pI or electrostatic charge influences the wash performance at various pH values.

(7) An embodiment of the invention relates to protease variants, wherein the net electrostatic charge has been changed in comparison to the parent protease at the same pH, and in which proteases there have, relative to said parent protease, either more positively charged amino acid residue(s) and/or fewer negatively charged amino acid residue(s), wherein protease variants comprising one or more of the alterations selected from the list consisting of: V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R wherein the positions corresponding to positions of SEQ ID NO 3.

(8) For those of the amino acids above having a negatively charged amino acid at the relevant pH replacing this amino acid with any neutral or positively charged amino acid at the same pH will result in an increased charge at that residue. According to one embodiment, the sum of amino acid charges of protease variants of the invention is greater than the sum of amino acid charges of the parent protease. Thus the protease variants of the invention have a net charge greater than that of the parent protease.

(9) Changing the net charge of SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5 to become larger in model B detergent (pH 7.8) increases the wash performance of the molecule as measured in Example 2 in Materials and Methods herein. The effect may be demonstrated by comparing with a set of variants engineered to become more negative under the same conditions.

(10) In one embodiment, the protease variants of the invention have increased net charge compared to the protease parent. One embodiment relates to a method of increasing the net charge of a protease having SEQ ID NO 3, wherein the method comprises a) introducing into a parent protease having SEQ ID NO 3 or having at least 60% identity hereto a substitution of an amino acids which is negative charged with an amino acid which is positively charged, neutral or with no amino acid i.e. deletion, wherein the substitutions/deletions are selected from the group consisting of the following: D17N, E50Q, HOT, E50L, D63N, D108S, D108A, D108N, D116S, D116A, D126G, D126W, D126K, E127Q, E127K, E127L, D147N, D147S, D147Q, D147H, D147W, D147*, D155H, E194*, E194S, E194K, E194Q, D206N, D206H, D218N, D218H, D218R, D225N, E236C, E279Q, E279H, D288N and D288K, wherein the variant has an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3; and b) recovering the variant.

(11) As described above, protease variants of the invention having an increased net charge compared to the protease parent have increased wash performance compared to the parent protease. In one embodiment the invention relates to protease variants having at least 60% identity to SEQ ID NO 3, having proteolytic activity and comprising one or more substitutions/deletions selected from the group consisting of: D17N, E50Q, HOT, E50L, D63N, D108S, D108A, D108N, D116S, D116A, D126G, D126W, D126K, E127Q, E127K, E127L, D147N, D147S, D147Q, D147H, D147W, D147*, D155H, E194*, E194S, E194K, E194Q, D206N, D206H, D218N, D218H, D218R, D225N, E236C, E279Q, E279H, D288N and D288K, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 3 and wherein the protease variants have improved wash performance compared to the parent or compared to SEQ ID NO 3. Wash performance may be measured as described in Example 2 herein. Another particular embodiment of the invention relates to a method of increasing the net charge of a protease having SEQ ID NO 3, wherein the method comprises c) introducing into a parent protease having SEQ ID NO 3 or having at least 60% identity hereto a substitution of an amino acids which is neutral or negative charged with an amino acid which is positively charged, wherein the substitutions are selected from the group consisting of the following substitutions: V2H, S4K, S4H, T5K, Q6K, N16K, N16H, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, Q57K, S58K, N59K, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, N109K, S111K, S111R, G112R, S114K, A118R, A118K, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, S148K, L149K, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, F207H, N212K, A214R, A214K, T215K, Y219R, Y219H, I220K, I220H, Q222K, A233R, S234K, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, V286H, V286K, Y287R, Y287H, I293R, I293K, T297K, D299R and K311R, wherein the variant has an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3; and d) recovering the variant.

(12) As described above, protease variants of the invention having an increased net charge compared to the protease parent have increased wash performance. In one embodiment the invention relates to protease variants having at least 60% identity to SEQ ID NO 3, having proteolytic activity and comprising one or more alterations selected from the group consisting of: V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 3 and wherein the protease variants have improved wash performance compared to the parent or compared to SEQ ID NO 3. Wash performance may be measured as described in Example 2 herein.

(13) A preferred embodiment of the invention relates to protease variants having at least 60% identity to SEQ ID NO 3, having proteolytic activity and comprising one or more alterations selected from the group consisting of: V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R, wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 3 and wherein the protease variants have an increased wash performance compared to the parent or compared to SEQ ID NO 3.

(14) In another aspect, the protease variant according to the invention comprises an alteration at two positions corresponding to any of positions 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297 or 311. In another aspect, a variant comprises an alteration at three positions corresponding to any of positions 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297 or 311.

(15) The present invention also relates to cleaning compositions such as detergent compositions comprising a protease variant of the invention. In one embodiment the cleaning composition is a liquid or powder laundry detergent, suitable for e.g. washing at high temperature and/or pH, such as at or above 40° C. and/or at or above pH 8. In one embodiment the cleaning composition is a liquid or powder laundry detergent, suitable for e.g. washing at low temperature and/or pH, such as at or below 20° C. and/or pH 6. The detergent may also be formulated as a unit dose detergent and/or compact detergent optionally with minimum or no water. The detergent may also be a dish wash detergent which is preferably phosphate-free. The cleaning composition may further comprise at least one additional enzyme, such as carbohydrate-active enzymes like carbohydrase, pectinase, mannanase, amylase, cellulase, arabinase, galactanase, xylanase, or proteases such as metalloproteases, lipase, a, cutinase, oxidase, e.g., a laccase, and/or peroxidase.

(16) In general positions within a protease useful for making variants are those positions wherein at least one substitution leads to a variant exhibiting an improved characteristic as compared to the unchanged protease The variants may further comprise one or more additional alterations at one or more (e.g., several) other positions. In a particular preferred embodiment the protease variants of the invention, further comprises a substitution at one or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO: 3, wherein the variant has a sequence identity to SEQ ID NO: 3 of at least 60%, and the variant has protease activity. In an even more preferred embodiment the amino acid at the position corresponding to position 171 of SEQ ID NO: 3 is selected from the group consisting of Trp, Lys, Glu, Asn and/or the amino acid at the position corresponding to position 173 of SEQ ID NO: 3 is Pro, and/or the amino acid at the position corresponding to position 175 of SEQ ID NO: 3 is Ala, Val, Pro, and/or the amino acid at the position corresponding to position 179 of SEQ ID NO: 3 is selected from the group consisting of Cys, Val, Gln, Ser, Thr, Glu, His, Lys, Met, Asn, Tyr and Ala and/or the amino acid at the position corresponding to position 180 of SEQ ID NO 3 is Tyr. In another preferred embodiment the protease variants of the invention, further comprises a substitution at two or more positions corresponding to positions 171, 173, 175, 179 or 180 of SEQ ID NO: 3, wherein the variant has a sequence identity to SEQ ID NO: 3 of at least 60% and less than 100%, and the variant has protease activity at two positions corresponding to any of positions 171, 173, 175, 179, and 180. In a preferred embodiment of the invention the variants of the invention comprises the mutations S173P+S175P as shown in SEQ ID NO 4. In another preferred embodiment the protease variants of the invention comprises the mutations S173P+S175P+F180Y as shown in SEQ ID NO 5. The protease variants of the invention may further comprise one or more of the substitutions: E50V, E50H, E127R, N199R or V286R.

(17) In a particular embodiment of the invention, the protease variants of the invention comprise the following alterations compared to SEQ ID NO 3: V2H, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S173P, S175P; S4K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, I137E, S173P, S175P, F180Y, T297P; A1*, V2*, P3*, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, N87K, E127Q, G132H, S173P, S175P, F180Y, Q198E, T297P; S4H, S173P, S175P; T5K, S173P, S175P; Q6K, S173P, S175P; N16K, S173P, S175P; N16K, D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, I137E, S173P, S175P, F180Y, T297P; D17N, S173P, S175P; D17N, I137E, S173P, S175P, F180Y, T297P; D17N, S173P, S175P, Q198E; D17N, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, I137E, S173P, S175P, F180Y, T297P; Q18K, S173P, S175P; Q18K, S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S19K, S173P, S175P; T24K, S173P, S175P; T24H, S173P, S175P; S27K, S173P, S175P; S27K, I137E, S173P, S175P, F180Y, T297P; S27K, S173P, S175P, Q198E; S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, S144R, S173P, S175P, F180Y, Q198E, T297P; S27K, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P; S27K, N87K, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P; D17N, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; N16H, S27K, S173P, S175P, F180Y, Q198E, T297P; S27K, I137E, S144R, S173P, S175P, F180Y, T297P; S27K, E127Q, I137E, S173P, S175P, F180Y, T297P; S27K, I137E, S173P, G174R, S175P, F180Y, T297P; S27K, Q97K, I137E, S173P, S175P, F180Y, T297P; D17N, S27K, I137E, S173P, S175P, F180Y, T297P; S27K, D126G, S173P, S175P, F180Y, T297P; Y39R, S173P, S175P; S27K, Y39R, S173P, S175P, F180Y, Q198E, T297P; S27K, Y39R, I137E, S173P, S175P, F180Y, T297P; S27K, Y39R, N87K, I137E, S173P, S175P, F180Y, T297P; S27K, Y39R, I137E, S144R, S173P, S175P, F180Y, T297P; S27K, Y39R, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S41K, S173P, S175P; S27K, S41K, E127Q, S173P, S175P, F180Y, Q198E, T297P; L43H, S173P, S175P; G47K, R130K, G132R, S173P, S175P; E50Q, S173P, S175P; E50Q, S173P, S175P, F180Y; E50Q, S173P, S175P, Q198E; E50Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P; E50T, S173P, S175P, F180Y; E50L, S173P, S175P, F180Y; G28N, Q57K, S86K, N87R, Q89R, S173P, S175P; S58K, S173P, S175P; S58K, S173P, S175P, F180Y, Q198E, T297P; N59K, S173P, S175P; D63N, S173P, S175P; D63E, R69D, S173P, S175P; G64H, S173P, S175P; S65K, S173P, S175P; S65K, S173P, S175P, F180Y, Q198E, T297P; S27K, S65K, S173P, S175P, F180Y, T297P; S27K, S65K, S173P, S175P, F180Y, T297P; T67K, S173P, S175P; Q70H, S173P, S175P, F180Y; G85R, S173P, S175P, F180Y; G85H, S173P, S175P, F180Y; S86K, S173P, S175P; N87K, S173P, S175P; N87K, I137E, S173P, S175P, F180Y, T297P; N87K, S173P, S175P, F180Y, Q198E, T297P; S27K, N87K, S173P, S175P, F180Y, Q198E, T297P; S27K, N87K, I137E, S173P, S175P, F180Y, T297P; S27K, N87K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P; S27K, N59L, T67V, N87K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N59L, T67V, N87K, G107N, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, Q70N, N87K, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, N199R, T297P; Y92R, S173P, S175P, F180Y; Q97K, S173P, S175P; Q97K, I137E, S173P, S175P, F180Y, T297P; Q97K, S173P, S175P, Q198E; Q97K, S173P, S175P, F180Y, Q198E, T297P; S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P; S27K, Q97K, I137E, S173P, S175P, F180Y, T297P; S27K, N59L, T67V, Q97K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; D108N, S173P, S175P; D108S, S173P, S175P; D108A, S173P, S175P; S27K, N109K, S173P, S175P, F180Y, Q198E, T297P; S111K, S173P, S175P; S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P; S114K, S173P, S175P, F180Y; D116S, M139L, S173P, S175P; A118R, S173P, S175P, F180Y; S27K, S111R, A118R, S173P, S175P, F180Y, Q198E, T297P; A118K, S173P, S175P, F180Y; D126G, S173P, S175P, F180Y; S27K, D126G, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P; D126W, S173P, S175P, F180Y; D126K, S173P, S175P, F180Y; Y113T, E127Q, S173P, G174K, S175P, F180Y; *109aS, E127Q, S173P, S175P, F180Y, T297P; E127Q, S173P, S175P, Q198E; E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, I137Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, S144R, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162S, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162E, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162Q, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162G, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162F, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162T, S173P, S175P, F180Y, Q198E, T297P; S27K, E127Q, V162Y, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P; S27K, N59L, T67V, N87K, G107N, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N87K, I121V, E127Q, S171N, S173P, S175P, F180Y, T297P; E127K, S173P, S175P, F180Y; E127L, S173P, S175P, F180Y; S129K, S173P, S175P; S41K, L43K, G47K, R130K, G132R, S173P, S175P; S41R, L43R, G47K, R130K, G132R, S173P, S175P; T131K, S173P, S175P; G132H, S173P, S175P; G132H, S173P, S175P, F180Y, Q198E, T297P; S143K, S173P, S175P; S144K, S173P, S175P; D147N, S173P, S175P; D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, D147N, S173P, S175P, F180Y, Q198E, T297P; S27K, N109S, S111R, D147N, S173P, S175P, F180Y, Q198E, T297P; D147S, S148A, L149T, I150L, S173P, S175P; A145S, K146P, D147S, S173P, S175P; A145S, K146P, D147S, S148A, S173P, S175P; S144P, A145S, K146P, D147S, S148A, L149T, I150L, S173P, S175P; D147S, L149T, S173P, S175P; K146P, D147S, S148A, L149T, I150L, S173P, S175P; A145S, K146P, D147S, S148A, L149T, S173P, S175P; S27K, K146E, D147S, S171N, S173P, G174R, S175P, F180Y, G182A, Q198E, N199K, E223K, T297P; K146*, D147*, S173P, S175P; D147Q, S173P, S175P; D147Q, S173P, S175P, F180Y, Q198E, T297P; D147H, S173P, S175P; S148K, S173P, S175P; L149K, S173P, S175P; D155H, S173P, S175P; Y156R, S173P, S175P; Y156R, S173P, S175P; Y156R, S173P, S175P; Y156R, S173P, S175P, Q198E; Y156R, S173P, S175P, F180Y, Q198E, T297P; S4K, D17N, S27K, N59L, N87K, Y156R, S173P, S175P, F180Y, Q198E, T297P; S4K, D17N, S27K, N59L, N87K, Y156R, S173P, S175P, F180Y, Q198E, T297P; Y156H, S173P, S175P; G159H, S173P, S175P; S27K, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, I137E, S173P, G174R, S175P, F180Y, T297P; S27K, N59L, T67V, N87K, S114V, I121V, I150L, S171N, S173P, G174R, S175P, F180Y, T297P; S173P, S175P, T177K; S173P, S175P, F180Y, L184H; I137E, S173P, S175P, F180Y, V185K, T297P; I137E, S173P, S175P, F180Y, V185K, N186P, T297P; I137E, S173P, S175P, F180Y, V185K, N186D, T297P; I137E, S173P, S175P, F180Y, N186K, T297P; S173P, S175P, N186K; I137E, S173P, S175P, F180Y, N186R, T297P; S173P, S175P, E194*; S173P, S175P, E194*, Q198E; S173P, S175P, F180Y, E194*, Q198E, T297P; S173P, S175P, F180Y, E194S; S27K, E127Q, S173P, S175P, F180Y, E194S, Q198E, T297P; S173P, S175P, F180Y, E194K; S27K, E127Q, S173P, S175P, F180Y, E194K, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, E194Q, Q198E, T297P; S173P, S175P, N195H; S27K, E127Q, S173P, S175P, F180Y, N195H, V196N, Q197*, Q198*, N199*, G200*, T201*, T297P; S173P, S175P, N195K; S173P, S175P, V196H; S173P, S175P, Q197K; S27K, S173P, S175P, F180Y, Q197K, T297P; S27K, S173P, S175P, F180Y, E194R, Q197K, Q198E, T297P; S27K, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, E127Q, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q197K, Q198E, T297P; S27K, S173P, G174K, S175P, F180Y, L184F, Q197K, Q198E, T297P; I121V, S173P, S175P, F180Y, Q197K, Q198E, T297P; S173P, S175P, Q197H; S173P, S175P, F180Y, Q197R; S27K, S171N, S173P, G174R, S175P, F180Y, Q197R, Q198E, T297P; S173P, S175P, Q198K; S173P, S175P, Q198K; S173P, S175P, N199K; S173P, S175P, N199K; S27K, S173P, S175P, F180Y, E194*, Q197K, Q198E, N199K, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, S173P, S175P, F180Y, Q197K, Q198E, N199K, T297P; S27K, Q70N, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S114V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, Q70N, G107N, I121V, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P; S27K, Q70A, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, Q70V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, S171R, S173P, G174E, S175P, F180Y, Q198E, N199K, T297P; S27K, S171K, S173P, G174E, S175P, F180Y, Q198E, N199K, T297P; S27K, S171E, S173P, G174K, S175P, F180Y, Q198E, N199K, T297P; S173P, S175P, G200H; S173P, S175P, D206N; S173P, S175P, N212K; S173P, S175P, F180Y, A214R; S173P, S175P, F180Y, A214K; S173P, S175P, T215K; S173P, S175P, D218N; S173P, S175P, D218H; S173P, S175P, F180Y, D218R; S173P, S175P, Y219R; S173P, S175P, Y219H; S173P, S175P, I220K; S173P, S175P, Q222K; S27K, Q97K, E127Q, S173P, S175P, F180Y, Q198E, D225N, I247M, T297P; S173P, S175P, F180Y, A233R; S173P, S175P, F180Y, A233R; S173P, S175P, F180Y, A233R; S173P, S175P, F180Y, A233R; S173P, S175P, F180Y, S234K; T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; A1*, P3*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P; S173P, S175P, T241K; S173P, S175P, N245K; S173P, S175P, T246K; S173P, S175P, S248K; S173P, S175P, F180Y, I264R; S173P, S175P, F180Y, I264R; S173P, S175P, F180Y, I264R; S173P, S175P, F180Y, I264R; S173P, S175P, A267H; S173P, S175P, N268K; S27K, E127Q, S173P, S175P, F180Y, Q198E, N268K, T297P; S173P, S175P, T269K; S173P, S175P, S270K; S173P, S175P, S270K; S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, S173P, S175P, F180Y, Q198E, S270K, T297P; S27K, I137E, S173P, S175P, F180Y, S270K, T297P; S27K, N87K, I121V, S171N, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, S270K, T297P; S27K, S173P, S175P, F180Y, S270K, T297P; S173P, S175P, S272K; S173P, S175P, S274K; S173P, S175P, T278K; S173P, S175P, E279Q; S173P, S175P, E279H; S173P, S175P, V286H; I137E, S173P, S175P, F180Y, V286K, T297P; I137E, S173P, S175P, F180Y, V286K, T297P; S173P, S175P, Y287R; S173P, S175P, Y287H; I137E, S173P, S175P, F180Y, D288N, T297P; S173P, S175P, F180Y, Q198E, D288N, T297P; I137E, S173P, S175P, F180Y, D288K, T297P; S27K, S173P, S175P, F180Y, Q198E, I293R, T297P; S27K, I137E, S173P, S175P, F180Y, I293R, T297P; S173P, S175P, I293K; S173P, S175P, T297K; G28D, S86K, N87R, Q89R, S173P, S175P, K311R; G28N, S86K, N87R, Q89R, S173P, S175P, K311R; E127S, S173P, S175P, F180Y; S173P, S175P, F180Y; S173P, S175P, F180Y, D299R; S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q197K, Q198E, S274V, T297P; S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, D147N, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P; S27K, D147S, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, G142E, D147N, I150S, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, G142E, I150S, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P; S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P; S27K, N109K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109K, S111D, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, N109K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109K, S111E, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, N109K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N109S, *109aE, S111R, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, N109S, *109aE, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, N87K, G132H, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, N87K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P; S27K, N87K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, N87K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, N87K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P; S27K, N87K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, Q70A, D147N, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, N109K, S111E, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, S111E, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S27K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S111D, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S111E, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P; S27K, S111R, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P; S27K, S114V, I121V, S129K, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P; S27K, S114V, I121V, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P; S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P; S27K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P; S27K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P; S27K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P; S4K, S27K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P; S4K, S27K, N87K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P and T24R, S173P, S175P, F180Y

(18) The variants may further comprise one or more additional alterations at one or more (e.g., several) other positions. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-5 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues, located at the amino- or carboxyl terminal; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

(19) Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Asn/Gln, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Glu/Gln, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

(20) Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

(21) Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for protease activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide. For the TY-145 protease (SEQ ID NO: 3) the catalytic triad comprising the amino acids D35, H72 and S251 is essential for protease activity of the enzyme.

(22) In an embodiment, the variant has improved catalytic activity compared to the parent enzyme.

(23) The homology between two amino acid sequences is in this context described by the parameter “identity” for purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as described above. The output from the routine is besides the amino acid alignment the calculation of the “Percent Identity” between the two sequences.

(24) Based on this description it is routine for a person skilled in the art to identify suitable homologous proteases, which can be modified according to the invention.

(25) Substantially homologous parent protease variants may have one or more (several) amino acid substitutions, deletions and/or insertions, in the present context the term “one or more” is used interchangeably with the term “several”. These changes are preferably of a minor nature, that is conservative amino acid substitutions as described above and other substitutions that do not significantly affect the three-dimensional folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag), such as a poly-histidine tract, or protein A (Nilsson et al., 1985, EMBO J. 4: 1075; Nilsson et al., 1991, Methods Enzymol. 198: 3. See, also, in general, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

(26) Although the changes described above preferably are of a minor nature, such changes may also be of a substantive nature such as fusion of larger polypeptides of up to 300 amino acids or more both as amino- or carboxyl-terminal extensions.

(27) The parent protease may comprise or consist of the amino acid sequence of SEQ ID NO: 3 or an allelic variant thereof; or a fragment thereof having protease activity. In one aspect, the parent protease comprises or consists of the amino acid sequence of SEQ ID NO: 3.

(28) The parent protease may be (a) a polypeptide having at least 60% sequence identity to polypeptide of SEQ ID NO: 3; (b) a polypeptide encoded by a polynucleotide that hybridizes under medium or high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i) or (ii); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

(29) In an aspect, the parent protease has a sequence identity to the polypeptide with SEQ ID NO: 3 of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% identity.

(30) In one aspect, the amino acid sequence of the parent protease differs by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the polypeptide with SEQ ID NO: 3.

(31) In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 3. In another aspect, the parent comprises or consists of amino acids 1 to 311 of SEQ ID NO: 2.

(32) In one aspect, the amino acid sequence of the parent protease differs by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the polypeptide with SEQ ID NO: 4.

(33) In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 4.

(34) In one aspect, the amino acid sequence of the parent protease differs by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the polypeptide with SEQ ID NO: 5.

(35) In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 5

(36) In another aspect, the parent protease is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, or high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) a sequence encoding the mature polypeptide of SEQ ID NO: 2, or (iii) the full-length complement of (i) or (ii), (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

(37) The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 3 or a fragment thereof may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed by the present invention.

(38) A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.

(39) For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) a sequence encoding the mature polypeptide of SEQ ID NO: 2; (iv) the full-length complement thereof; or (v) a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

(40) In one aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another aspect, the nucleotide acid probe is a 80 to 1140 nucleotides long fragment of SEQ ID NO: 1, e.g. 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or 1100 nucleotides long. In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2.

(41) In another embodiment, the parent is encoded by a polynucleotide having a sequence identity of at least 60%, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to the mature polypeptide coding sequence of SEQ ID NO: 1 or a sequence encoding the mature polypeptide of SEQ ID NO: 2.

(42) The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

(43) The parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

(44) A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

(45) The parent may be obtained from organisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

(46) The parent may be a bacterial protease. For example, the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces protease, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma protease.

(47) In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausfi, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis protease

(48) In one aspect, the parent is a Bacillus sp. protease, e.g., the protease with SEQ ID NO: 3 or the mature polypeptide of SEQ ID NO 2.

(49) Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

(50) The parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

(51) Preparation of Variants

(52) The present invention also relates to a method for obtaining a protease variant having at least one improved property compared to SEQ ID NO 3, comprising e) introducing into a parent protease having at least 60% identity to of SEQ ID NO: 3 one or more of the following alterations: V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R, wherein the variant has an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3; and f) recovering the variant.

(53) The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

(54) The present invention relates to a method for obtaining a protease variant having at least one improved property compared to SEQ ID NO 3, comprising introducing into a parent protease with at least 65% identity to SEQ ID NO: 3 a substitution at one or more positions: 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297 or 311 corresponding to positions of SEQ ID NO 3 wherein the variant has an amino acid sequence which is at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3; and recovering the variant.

(55) The present invention relates to a method for obtaining a protease variant having at least one improved property compared to SEQ ID NO 3, comprising introducing into a parent protease with at least 75% identity to SEQ ID NO: 3 a substitution at two or more positions: 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297 or 311 corresponding to positions of SEQ ID NO 3 wherein the variant has an amino acid sequence which is at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3; and recovering the variant.

(56) The variants of the invention may also be prepared by procedures such as those mentioned below.

(57) Site-directed mutagenesis is a technique in which one or more (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

(58) Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

(59) Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

(60) Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432: 1050-1054) and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

(61) Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

(62) Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

(63) Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

(64) Polynucleotides

(65) The present invention also relates to isolated polynucleotides encoding a variant of the present invention.

(66) Nucleic Acid Constructs

(67) The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

(68) The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

(69) The control sequence may be a promoter, a polynucleotide which is recognized by a host cell for expression of the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

(70) Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in “Useful proteins from recombinant bacteria” in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

(71) The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.

(72) Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

(73) The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

(74) Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

(75) The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell's secretory pathway. The 5′-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5′-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

(76) Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

(77) The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

(78) Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of the variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

(79) It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.

(80) Expression Vectors

(81) The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

(82) The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

(83) The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

(84) The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

(85) Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance.

(86) The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

(87) For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

(88) For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

(89) Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMß1 permitting replication in Bacillus.

(90) More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a variant. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

(91) The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

(92) Host Cells

(93) The present invention also relates to recombinant host cells, comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.

(94) The host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.

(95) The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

(96) The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

(97) The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

(98) The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

(99) The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

(100) Methods of Production

(101) The present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant.

(102) The host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the variant to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

(103) The variant may be detected using methods known in the art that are specific for the variants with protease activity. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant.

(104) The variant may be recovered using methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

(105) The variant may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

(106) In an alternative aspect, the variant is not recovered, but rather a host cell of the present invention expressing the variant is used as a source of the variant.

(107) Compositions

(108) In one certain aspect, the variants according to the invention has improved stability in detergents compared to the parent enzyme or compared to a protease having the identical amino acid sequence of said variant but not having the alterations at one or more of said specified positions or compared to a protease with SEQ ID NO 3, wherein stability is measured in Example 2 as described in “Material and Methods” herein.

(109) Besides enzymes the detergent compositions may comprise additional components. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.

(110) Detergent Compositions of the Present Invention

(111) The variants of the present invention may be added to a detergent composition in an amount corresponding to 0.001-100 mg of protein, such as 0.01-100 mg of protein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein per liter of wash liquor.

(112) The variants of the present invention may be stabilized using stabilizing agents, which may be selected from the group containing propylene glycol, glycerol, a sugar, a sugar alcohol, lactic acid, boric acid, borate and phenyl boronic acid derivates, such as 4-formylphenylboronic acid (4-FPBA).

(113) The variants according to the invention may also be stabilized using peptide aldehydes or ketones such as described in WO 2005/105826 and WO 2009/118375. A variant of the present invention may also be incorporated in the detergent formulations disclosed in WO 97/07202, which is hereby incorporated by reference.

(114) Surfactants

(115) The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from about 0.1% to 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized.

(116) When included therein, the detergent will usually contain from about 1% to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

(117) When included therein, the detergent will usually contain from about 1% to about 40% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylehanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, and combinations thereof, Alkyl quaternary ammonium compounds, Alkoxylated quaternary ammonium (AQA),

(118) When included therein, the detergent will usually contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

(119) When included therein, the detergent will usually contain from about 1% to about 40% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

(120) When included therein, the detergent will usually contain from about 1% to about 40% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, and sulfobetaine, and combinations thereof.

(121) Hydrotropes

(122) A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

(123) The detergent may contain 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonates (STS), sodium xylene sulfonates (SXS), sodium cumene sulfonates (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

(124) Builders and Co-Builders

(125) The detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co-builder known in the art for use in laundry, ADW and hard surfaces cleaning detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA) and 2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin (CMI), and combinations thereof.

(126) The detergent composition may also contain 0-65% by weight, such as about 5% to about 40%, of a detergent co-builder, or a mixture thereof. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include 2,2′,2″-nitrilotriacetic acid (NTA), etheylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N′-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonic acid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl) aspartic acid (SEAS), N-(2-sulfomethyl) glutamic acid (SMGL), N-(2-sulfoethyl) glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylidenediaminetriacetate (HEDTA), diethanolglycine (DEG), Diethylenetriamine Penta (Methylene Phosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854, U.S. Pat. No. 5,977,053.

(127) Bleaching Systems

(128) The detergent may contain 0-10% by weight, such as about 1% to about 5%, of a bleaching system. Any bleaching system known in the art for use in laundry, ADW and hard surfaces cleaning detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone®, and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator. By bleach activator is meant herein a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides. Suitable examples are tetracetyl ethylene diamine (TAED), sodium 3,5,5 trimethyl hexanoyloxybenzene sulphonat, diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS), tetraacetylethylenediamine (TAED) and 4-(nonanoyloxy)benzenesulfonate (NOBS), and/or those disclosed in WO98/17767. A particular family of bleach activators of interest was disclosed in EP624154 and particulary preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like Triacin has the advantage that it is environmental friendly as it eventually degrades into citric acid and alcohol. Furthermore acethyl triethyl citrate and triacetin has a good hydrolytical stability in the product upon storage and it is an efficient bleach activator. Finally ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6-(phthaloylamino)percapronic acid (PAP). The bleaching system may also include a bleach catalyst. In some embodiments the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formulae:

(129) ##STR00001##
(iii) and mixtures thereof; wherein each R.sup.1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R.sup.1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R.sup.1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described, e.g., in WO2007/087258, WO2007/087244, WO2007/087259, WO2007/087242. Suitable photobleaches may for example be sulfonated zinc phthalocyanine
Polymers

(130) The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of polyethylene terephthalate and polyoxyethene terephthalate (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridin-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.

(131) Fabric Hueing Agents

(132) The detergent compositions may also include fabric hueing agents such as dyes or pigments which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liquor comprising said detergent compositions thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby incorporated by reference). A detergent composition preferably comprises from about 0.00003 wt % to about 0.2 wt %, from about 0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % to about 0.04 wt % fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g., WO 2007/087257, WO2007/087243.

(133) Additional Enzymes

(134) In one embodiment, the variants according to the invention are combined with one or more enzymes, such as at least two enzymes, more preferred at least three, four or five enzymes. Preferably, the enzymes have different substrate specificity, e.g., proteolytic activity, amylolytic activity, lipolytic activity, hemicellulytic activity or pectolytic activity.

(135) The detergent additive as well as the detergent composition may comprise one or more additional enzymes such as carbohydrate-active enzymes like carbohydrase, pectinase, mannanase, amylase, cellulase, arabinase, galactanase, xylanase, or protease, lipase, a, cutinase, oxidase, e.g., a laccase, and/or peroxidase.

(136) In general, the properties of the selected enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.

(137) Cellulases:

(138) Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757 and WO 89/09259.

(139) Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 and WO99/001544.

(140) Other cellulases are endo-beta-1,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.

(141) Commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes NS) Carezyme Premium™ (Novozymes A/S), Celluclean™ (Novozymes A/S), Celluclean Classic™ (Novozymes A/S), Cellusoft™ (Novozymes A/S), Whitezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

(142) Mannanases

(143) Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes A/S).

(144) Proteases:

(145) Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4 or other metalloprotease such as those from M5, M7 or M8 families.

(146) The term “subtilases” refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.

(147) Examples of subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140). Other useful proteases may be those described in WO92/175177, WO01/016285, WO02/026024 and WO02/016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270, WO94/25583 and WO05/040372, and the chymotrypsin proteases derived from Cellulomonas described in WO05/052161 and WO05/052146.

(148) A further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in WO95/23221, and variants thereof which are described in WO92/21760, WO95/23221, EP1921147 and EP1921148.

(149) Examples of metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int.) such as those derived from Bacillus amyloliquefaciens.

(150) Examples of useful proteases are the variants described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263, WO11/036264, especially the variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred the protease variants may comprise the mutations: S3T, V41, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, 5130A, G160D, Y167A, R1705, A194P, G195E, V199M, V2051, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

(151) Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, BLAZE®, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequence shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

(152) Lipases and Cutinases:

(153) Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipase from Thermobifida fusca (WO11/084412), Geobacillus stearothermophilus lipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (WO12/137147).

(154) Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.

(155) Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

(156) Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143), acyltransferase from Mycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variants of the M. smegmatis perhydrolase in particular the 554V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

(157) Amylases:

(158) Suitable amylases which can be used together with the variants of the invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839.

(159) Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 or variants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

(160) Different suitable amylases include amylases having SEQ ID NO: 6 in WO 02/010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

(161) Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90% sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having the substitutions:

(162) M197T;

(163) H156Y+A181T+N190F+A209V+Q264S; or

(164) G48A+T491+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

(165) Further amylases which are suitable are amylases having SEQ ID NO: 6 in WO 99/019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.

(166) Additional amylases which can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/023873 for numbering. More preferred variants are those having a deletion in two positions selected from 181, 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

(167) Other amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

(168) Further suitable amylases are amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90% sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181 or of T182 and/or G183. Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:

(169) N128C+K178L+T182G+Y305R+G475K;

(170) N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

(171) S125A+N128C+K178L+T182G+Y305R+G475K; or

(172) S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants are C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.

(173) Further suitable amylases are amylases having SEQ ID NO: 1 of WO13184577 or variants having 90% sequence identity to SEQ ID NO: 1 thereof. Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or deletion in position R178 and/or S179 or of T180 and/or G181. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:

(174) E187P+I203Y+G476K

(175) E187P+I203Y+R458N+T459S+D460T+G476K,

(176) wherein the variants optionally further comprises a substitution at position 241 and/or a deletion at position 178 and/or position 179.

(177) Further suitable amylases are amylases having SEQ ID NO: 1 of WO10104675 or variants having 90% sequence identity to SEQ ID NO: 1 thereof. Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: N21, D97, V128 K177, R179, S180, I181, G182, M200, L204, E242, G477 and G478. More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: N21D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion in position R179 and/or S180 or of 1181 and/or G182. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:

(178) N21D+D97N+V128I,

(179) wherein the variants optionally further comprises a substitution at position 200 and/or a deletion at position 180 and/or position 181.

(180) Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.

(181) Other examples are amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.

(182) Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A/S), and Rapidase™ Purastar™/Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc./DuPont).

(183) Peroxidases/Oxidases:

(184) Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.

(185) Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

(186) Other Enzymes:

(187) A protease variant according to the invention may also be combined with additional enzymes such as pectate lyases e.g. Pectawash™, chlorophyllases etc. The protease variant of the invention may be mixed with any additional enzyme.

(188) The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive, i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

(189) Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.

(190) Adjunct Materials

(191) Any detergent components known in the art for use in laundry detergents may be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

(192) Dispersants

(193) The detergent compositions can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.

(194) Dye Transfer Inhibiting Agents—

(195) The detergent compositions may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the composition.

(196) Fluorescent Whitening Agent—

(197) A detergent compositions will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene-2,2′-disulphonate; 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2′-disulphonate; 4,4′-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2′-disulphonate, 4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2′-disulphonate; 4,4′-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2′-disulphonate and 2-(stilbyl-4″-naptho-1,2′:4,5)-1,2,3-trizole-2″-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4′-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2′-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use include the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.

(198) Suitable fluorescent brightener levels include lower levels of from about 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.

(199) Soil Release Polymers—

(200) The detergent composition may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalte based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference). Furthermore random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose deriviatives such as those described in EP 1867808 or WO 2003/040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.

(201) Anti-Redeposition Agents—

(202) The detergent compositions may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.

(203) Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, structurants for liquid detergents and/or structure elasticizing agents.
Formulation of Detergent Products

(204) The detergent composition may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.

(205) Detergent formulation forms: Layers (same or different phases), Pouches, versus forms for Machine dosing unit.

(206) Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxyprpyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blend compositions comprising hydrolytically degradable and water soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticisers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water soluble film. The compartment for liquid components can be different in composition than compartments containing solids. Ref: (US2009/0011970 A1)

(207) Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

(208) Definition/Characteristics of the Forms:

(209) A liquid or gel detergent, which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent.

(210) A liquid or gel detergent may be non-aqueous.

(211) Methods and Uses

(212) The protease variants of the present invention may be added to and thus become a component of a detergent composition, wherein said variant comprises an alteration at one or more positions corresponding to positions 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297 or 311 of SEQ ID NO: 3, wherein the variant has at least 60%, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 3. Detergent compositions is generally used in cleaning processes such as laundry and/or hard surface cleaning e.g. dish wash.

(213) One embodiment of the invention relates a detergent composition, such as a laundry or dish wash compositions comprising a protease variant of a protease parent having at least 60% identity to SEQ ID NO 3 wherein said variant comprises at least one alterations compared to said parent protease at an amino acid occupying any of the positions corresponding to positions: 2, 4, 5, 6, 16, 17, 18, 19, 24, 27, 39, 41, 43, 47, 50, 57, 58, 59, 63, 64, 65, 67, 70, 85, 86, 87, 92, 97, 108, 109, 111, 112, 114, 116, 118, 126, 127, 129, 130, 131, 132, 142, 143, 144, 147, 148, 149, 155, 156, 174, 177, 184, 185, 186, 194, 195, 196, 197, 198, 199, 200, 206, 207, 212, 214, 215, 218, 219, 220, 222, 225, 234, 236, 241, 245, 246, 247, 248, 264, 267, 268, 269, 270, 272, 274, 278, 279, 286, 287, 288, 293, 297 or 311 of SEQ ID NO 3, wherein the variant has an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 3.

(214) A detergent composition may comprise at least one a variant wherein said variant comprises one or more of the following alterations: V2H, S4K, S4H, T5K, Q6K, N16K, N16H, D17N, Q18K, Q18H, S19K, T24K, T24H, T24R, S27K, Y39R, S41K, L43H, G47K, E50Q, E50T, E50L, Q57K, S58K, N59K, D63N, G64H, S65K, T67K, Q70H, G85R, G85H, S86K, N87K, Y92R, Q97K, D108N, D108S, D108A, N109K, S111K, S111R, G112R, S114K, D116S, D116A, A118R, A118K, D126G, D126W, D126K, E127Q, E127K, E127L, E127S, S129K, R130K, T131K, T131H, G132H, G142K, G142R, S143K, S143H, S144K, D147N, D147S, D147*, D147Q, D147H, D147W, S148K, L149K, D155H, Y156R, Y156H, G159H, G174R, G174H, T177K, L184H, V185K, N186K, N186R, E194*, E194S, E194K, E194Q, N195H, N195K, V196H, Q197K, Q197H, Q197R, Q198K, N199K, G200H, D206N, D206H, F207H, N212K, A214R, A214K, T215K, D218N, D218H, D218R, Y219R, Y219H, I220K, I220H, Q222K, D225N, A233R, S234K, E236C, T241K, N245K, T246K, I247K, S248K, I264R, A267H, N268K, N268H, T269K, T269H, S270K, S272K, S274K, T278K, T278H, E279Q, E279H, V286H, V286K, Y287R, Y287H, D288N, D288K, I293R, I293K, T297K, D299R and K311R of SEQ ID NO: 3, wherein the variant has a sequence identity to SEQ ID NO: 3 of at least 60% such as at least at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 3 and the variant has protease activity. The at least one protease variant preferably has increased detergent stability relative to the parent or relative to a protease parent having the identical amino acid sequence of said variant but not having the substitutions at one or more of said positions when tested in Assay B described in example 2, as described under “Material and Methods”.

(215) A detergent composition may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

(216) A cleaning process or the textile care process may for example be a laundry process, a dishwashing process or cleaning of hard surfaces such as bathroom tiles, floors, table tops, drains, sinks and washbasins. Laundry processes can for example be household laundering, but it may also be industrial laundering. A process for laundering of fabrics and/or garments may be a process comprises treating fabrics with a washing solution containing a detergent composition, and at least one protease variant. A cleaning process or a textile care process can for example be carried out in a machine washing process or in a manual washing process. The washing solution can for example be an aqueous washing solution containing a detergent composition.

(217) The fabrics and/or garments subjected to a washing, cleaning or textile care process may be conventional washable laundry, for example household laundry. Preferably, the major part of the laundry is garments and fabrics, including knits, woven, denims, non-woven, felts, yarns, and towelling. The fabrics may be cellulose based such as natural cellulosics, including cotton, flax, linen, jute, ramie, sisal or coir or manmade cellulosics (e.g., originating from wood pulp) including viscose/rayon, ramie, cellulose acetate fibers (tricell), lyocell or blends thereof. The fabrics may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabit and silk or synthetic polymer such as nylon, aramid, polyester, acrylic, polypropylen and spandex/elastane, or blends thereof as well as blend of cellulose based and non-cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., rayon/viscose, ramie, flax, linen, jute, cellulose acetate fibers, lyocell).

(218) The last few years there has been an increasing interest in replacing components in detergents, which is derived from petrochemicals with renewable biological components such as enzymes and polypeptides without compromising the wash performance. When the components of detergent compositions change new enzyme activities or new enzymes having alternative and/or improved properties compared to the common used detergent enzymes such as proteases, lipases and amylases is needed to achieve a similar or improved wash performance when compared to the traditional detergent compositions.

(219) Proteases and variants hereof are usable in proteinaceous stain removing processes. The proteinaceous stains may be stains such as food stains, e.g., baby food, sebum, cocoa, egg, blood, milk, ink, grass, or a combination hereof.

(220) Typical detergent compositions include various components in addition to the enzymes, these components have different effects, some components like the surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away, other components like bleach systems remove discolor often by oxidation and many bleaches also have strong bactericidal properties, and are used for disinfecting and sterilizing. Yet other components like builder and chelator softens, e.g., the wash water by removing the metal ions form the liquid.

(221) The enzyme compositions may further comprise at least one or more of the following: a surfactant, a builder, a chelator or chelating agent, bleach system or bleach component in laundry or dish wash.

(222) The amount of a surfactant, a builder, a chelator or chelating agent, bleach system and/or bleach component may be reduced compared to amount of surfactant, builder, chelator or chelating agent, bleach system and/or bleach component used without the added protease variant of the invention. Preferably the at least one component which is a surfactant, a builder, a chelator or chelating agent, bleach system and/or bleach component is present in an amount that is 1% less, such as 2% less, such as 3% less, such as 4% less, such as 5% less, such as 6% less, such as 7% less, such as 8% less, such as 9% less, such as 10% less, such as 15% less, such as 20% less, such as 25% less, such as 30% less, such as 35% less, such as 40% less, such as 45% less, such as 50% less than the amount of the component in the system without the addition of protease variants of the invention, such as a conventional amount of such component. Detergent compositions may also be composition which is free of at least one component which is a surfactant, a builder, a chelator or chelating agent, bleach system or bleach component and/or polymer.

(223) Washing Method

(224) Detergent compositions are ideally suited for use in laundry applications. These methods include a method for laundering a fabric. The method comprises the steps of contacting a fabric to be laundered with a cleaning laundry solution comprising a detergent composition. The fabric may comprise any fabric capable of being laundered in normal consumer use conditions. The solution preferably has a pH from about 5.5 to about 11.5. The compositions may be employed at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. The water temperatures typically range from about 5° C. to about 95° C., including about 10° C., about 15° C., about 20° C., about 25° C., about 30° C., about 35° C., about 40° C., about 45° C., about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., about 75° C., about 80° C., about 85° C. and about 90° C. The water to fabric ratio is typically from about 1:1 to about 30:1.

(225) In particular embodiments, the washing method is conducted at a pH from about 5.0 to about 11.5, or from about 6 to about 10.5, about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5. to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to about 8, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 11, about 9 to about 10, about 10 to about 11, preferably about 5.5 to about 11,5.

(226) In particular embodiments, the washing method is conducted at a degree of hardness of from about 0° dH to about 30° dH, such as about 1° dH, about 2° dH, about 3° dH, about 4° dH, about 5° dH, about 6° dH, about 7° dH, about 8° dH, about 9° dH, about 10° dH, about 11° dH, about 12° dH, about 13° dH, about 14° dH, about 15° dH, about 16° dH, about 17° dH, about 18° dH, about 19° dH, about 20° dH, about 21° dH, about 22° dH, about 23° dH, about 24° dH, about 25° dH, about 26° dH, about 27° dH, about 28° dH, about 29° dH, about 30° dH. Under typical European wash conditions, the degree of hardness is about 16° dH, under typical US wash conditions about 6° dH, and under typical Asian wash conditions, about 3° dH.

(227) The compositions for use in the methods described above may further comprises at least one additional enzyme as set forth in the “other enzymes” section above, such as an enzyme selected from the group of hydrolases such as proteases, lipases and cutinases, carbohydrases such as amylases, cellulases, hemicellulases, xylanases, and pectinase or a combination hereof.

(228) The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

(229) Materials and Methods

(230) Protease Activity Assays

(231) 1) Suc-AAPF-pNA Activity Assay:

(232) The proteolytic activity can be determined by a method employing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-Succinyl-Alanine-Alanine-Proline-Phenylalanine-p-Nitroanilide, and it is a blocked peptide which can be cleaved by endo-proteases. Following cleavage a free pNA molecule is liberated and it has a yellow colour and thus can be measured by visible spectrophotometry at wavelength 405 nm. The Suc-AAPF-PNA substrate is manufactured by Bachem (cat. no. L1400, dissolved in DMSO).

(233) The protease sample to be analyzed was diluted in residual activity buffer (100 mM Tris pH 8.6). The assay was performed by transferring 30 μl of diluted enzyme samples to 96 well microtiter plate and adding 70 μl substrate working solution (0.72 mg/ml in 100 mM Tris pH8.6). The solution was mixed at room temperature and absorption is measured every 20 sec. over 5 minutes at OD 405 nm.

(234) The slope (absorbance per minute) of the time dependent absorption-curve is directly proportional to the activity of the protease in question under the given set of conditions. The protease sample should be diluted to a level where the slope is linear.

(235) TABLE-US-00001 TABLE 1 Composition of model detergent B Ingredient wt % (C10-C13)alkylbenzene sulfonic acid 7.2 sodium lauryl ether sulfate 10.6 cocoa fatty acid 2.75 soy fatty acid 2.75 alcohol ethoxylate 6.6 sodium hydroxide 1.1 Ethanol 3 propane-1,2-diol 6 Glycerol 1.7 Triethanolamine 3.3 sodium formiate 1 sodium citrate 2 diethylenetriaminepentakis(methylene)pentakis(phosphonic acid) 0.5 copoly(acrylic acid/maleic acid) 0.5 ion-exchanged water 51
General Molecular Biology Methods:

(236) Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989); Ausubel et al. (1995); Harwood and Cutting (1990).

Example 1: Construction of TY145 Variants by Site-Directed Mutagenesis

(237) Site-directed variants were constructed of the TY145 protease (SEQ ID NO: 3) comprising specific substitutions according to the invention. The variants were made by traditional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) using PCR together with properly designed mutagenic oligonucleotides that introduced the desired mutations in the resulting sequence.

(238) Mutagenic oligos were designed corresponding to the DNA sequence flanking the desired site(s) of mutation, separated by the DNA base pairs defining the insertions/deletions/substitutions, and purchased from an oligo vendor such as Life Technologies. In this manner, the variants listed in Table 2 below were constructed and produced.

(239) In order to test TY145 protease variants of the invention, the mutated DNA comprising a variant of the invention was transformed into a competent B. subtilis strain and fermented using standard protocols (liquid media, 3-4 days, 30° C.). The culture broth was centrifuged (26000×g, 20 min) and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filtration unit in order to remove the rest of the Bacillus host cells. The 0.2 μm filtrate was mixed 1:1 with 3.0M (NH4)2SO4 and the mixture was applied to a Phenyl-sepharose FF (high sub) column (from GE Healthcare) equilibrated in 100 mM H3BO3, 10 mM MES/NaOH, 2 mM CaCl2, 1.5M (NH4)2SO4, pH 6.0. After washing the column with the equilibration buffer, the protease was step-eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, pH 6.0. The eluted peak (containing the protease activity) was collected and applied to a Bacitracin agarose column (from Upfront chromatography) equilibrated in 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, pH 6.0. After washing the column extensively with the equilibration buffer, the protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, 1M NaCl, pH 6.0 with 25% (v/v) 2-propanol. The elution peak (containing the protease activity) was transferred to 20 mM MES, 2 mM CaCl2, pH 6.0 on a G25 sephadex column (from GE Healthcare). The G25 transferred peak was the purified preparation and was used for further experiments.

(240) TABLE-US-00002 TABLE 2 Variants of SEQ ID NO 3 a - V2H, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P S4K, S173P, S175P S4K, S173P, S175P, F180Y, Q198E, T297P S4K, S27K, S173P, S175P, F180Y, Q198E, T297P S4K, S27K, I137E, S173P, S175P, F180Y, T297P A1*, V2*, P3*, S4K, S27K, S173P, S175P, F180Y, Q198E, T297P S4K, S27K, N87K, E127Q, G132H, S173P, S175P, F180Y, Q198E, T297P S4H, S173P, S175P T5K, S173P, S175P Q6K, S173P, S175P N16K, S173P, S175P N16K, D17N, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, I137E, S173P, S175P, F180Y, T297P D17N, S173P, S175P D17N, I137E, S173P, S175P, F180Y, T297P D17N, S173P, S175P, Q198E D17N, S173P, S175P, F180Y, Q198E, T297P D17N, S27K, S173P, S175P, F180Y, Q198E, T297P D17N, S27K, I137E, S173P, S175P, F180Y, T297P Q18K, S173P, S175P Q18K, S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P S19K, S173P, S175P T24K, S173P, S175P T24H, S173P, S175P S27K, S173P, S175P S27K, I137E, S173P, S175P, F180Y, T297P S27K, S173P, S175P, Q198E S27K, S173P, S175P, F180Y, Q198E, T297P S27K, S144R, S173P, S175P, F180Y, Q198E, T297P S27K, D147N, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P S27K, N87K, S173P, S175P, F180Y, Q198E, T297P S4K, S27K, S173P, S175P, F180Y, Q198E, T297P S27K, S173P, S175P, F180Y, Q198E, S270K, T297P S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P D17N, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, S173P, S175P, F180Y, Q198E, T297P N16H, S27K, S173P, S175P, F180Y, Q198E, T297P S27K, I137E, S144R, S173P, S175P, F180Y, T297P S27K, E127Q, I137E, S173P, S175P, F180Y, T297P S27K, I137E, S173P, G174R, S175P, F180Y, T297P S27K, Q97K, I137E, S173P, S175P, F180Y, T297P D17N, S27K, I137E, S173P, S175P, F180Y, T297P S27K, D126G, S173P, S175P, F180Y, T297P Y39R, S173P, S175P S27K, Y39R, S173P, S175P, F180Y, Q198E, T297P S27K, Y39R, I137E, S173P, S175P, F180Y, T297P S27K, Y39R, N87K, I137E, S173P, S175P, F180Y, T297P S27K, Y39R, I137E, S144R, S173P, S175P, F180Y, T297P S27K, Y39R, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P S41K, S173P, S175P S27K, S41K, E127Q, S173P, S175P, F180Y, Q198E, T297P L43H, S173P, S175P G47K, R130K, G132R, S173P, S175P E50Q, S173P, S175P E50Q, S173P, S175P, F180Y E50Q, S173P, S175P, Q198E E50Q, S173P, S175P, F180Y, Q198E, T297P S27K, E50Q, S173P, S175P, F180Y, Q198E, T297P E50T, S173P, S175P, F180Y E50L, S173P, S175P, F180Y G28N, Q57K, S86K, N87R, Q89R, S173P, S175P S58K, S173P, S175P S58K, S173P, S175P, F180Y, Q198E, T297P N59K, S173P, S175P D63N, S173P, S175P D63E, R69D, S173P, S175P G64H, S173P, S175P S65K, S173P, S175P S65K, S173P, S175P, F180Y, Q198E, T297P S27K, S65K, S173P, S175P, F180Y, T297P S27K, S65K, S173P, S175P, F180Y, T297P T67K, S173P, S175P Q70H, S173P, S175P, F180Y G85R, S173P, S175P, F180Y G85H, S173P, S175P, F180Y S86K, S173P, S175P N87K, S173P, S175P N87K, I137E, S173P, S175P, F180Y, T297P N87K, S173P, S175P, F180Y, Q198E, T297P S27K, N87K, S173P, S175P, F180Y, Q198E, T297P S27K, N87K, I137E, S173P, S175P, F180Y, T297P S27K, N87K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P S27K, N59L, T67V, N87K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, N59L, T67V, N87K, G107N, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, Q70N, N87K, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, N199R, T297P Y92R, S173P, S175P, F180Y Q97K, S173P, S175P Q97K, I137E, S173P, S175P, F180Y, T297P Q97K, S173P, S175P, Q198E Q97K, S173P, S175P, F180Y, Q198E, T297P S27K, Q97K, S173P, S175P, F180Y, Q198E, T297P S27K, Q97K, I137E, S173P, S175P, F180Y, T297P S27K, N59L, T67V, Q97K, G107N, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P D108N, S173P, S175P D108S, S173P, S175P D108A, S173P, S175P S27K, N109K, S173P, S175P, F180Y, Q198E, T297P S111K, S173P, S175P S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, T297P S114K, S173P, S175P, F180Y D116S, M139L, S173P, S175P A118R, S173P, S175P, F180Y S27K, S111R, A118R, S173P, S175P, F180Y, Q198E, T297P A118K, S173P, S175P, F180Y D126G, S173P, S175P, F180Y S27K, D126G, S173P, S175P, F180Y, Q198E, T297P S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P D126W, S173P, S175P, F180Y D126K, S173P, S175P, F180Y Y113T, E127Q, S173P, G174K, S175P, F180Y *109aS, E127Q, S173P, S175P, F180Y, T297P E127Q, S173P, S175P, Q198E E127Q, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, S173P, S175P, F180Y, Q198E, T297P S27K, D126G, E127Q, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, I137Q, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, S144R, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162S, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162E, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162Q, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162G, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162F, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162T, S173P, S175P, F180Y, Q198E, T297P S27K, E127Q, V162Y, S173P, S175P, F180Y, Q198E, T297P S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P S27K, N109S, S111R, E127Q, S173P, S175P, F180Y, Q198E, T297P S27K, N59L, T67V, N87K, G107N, I121V, E127Q, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, N87K, I121V, E127Q, S171N, S173P, S175P, F180Y, T297P E127K, S173P, S175P, F180Y E127L, S173P, S175P, F180Y S129K, S173P, S175P S41K, L43K, G47K, R130K, G132R, S173P, S175P S41R, L43R, G47K, R130K, G132R, S173P, S175P T131K, S173P, S175P G132H, S173P, S175P G132H, S173P, S175P, F180Y, Q198E, T297P S143K, S173P, S175P S144K, S173P, S175P D147N, S173P, S175P D147N, S173P, S175P, F180Y, Q198E, T297P S27K, D147N, S173P, S175P, F180Y, Q198E, T297P S27K, N109S, S111R, D147N, S173P, S175P, F180Y, Q198E, T297P D147S, S148A, L149T, I150L, S173P, S175P A145S, K146P, D147S, S173P, S175P A145S, K146P, D147S, S148A, S173P, S175P S144P, A145S, K146P, D147S, S148A, L149T, I150L, S173P, S175P D147S, L149T, S173P, S175P K146P, D147S, S148A, L149T, I150L, S173P, S175P A145S, K146P, D147S, S148A, L149T, S173P, S175P S27K, K146E, D147S, S171N, S173P, G174R, S175P, F180Y, G182A, Q198E, N199K, E223K, T297P K146*, D147*, S173P, S175P D147Q, S173P, S175P D147Q, S173P, S175P, F180Y, Q198E, T297P D147H, S173P, S175P S148K, S173P, S175P L149K, S173P, S175P D155H, S173P, S175P Y156R, S173P, S175P Y156R, S173P, S175P Y156R, S173P, S175P Y156R, S173P, S175P, Q198E Y156R, S173P, S175P, F180Y, Q198E, T297P S4K, D17N, S27K, N59L, N87K, Y156R, S173P, S175P, F180Y, Q198E, T297P S4K, D17N, S27K, N59L, N87K, Y156R, S173P, S175P, F180Y, Q198E, T297P Y156H, S173P, S175P G159H, S173P, S175P S27K, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, I137E, S173P, G174R, S175P, F180Y, T297P S27K, N59L, T67V, N87K, S114V, I121V, I150L, S171N, S173P, G174R, S175P, F180Y, T297P S173P, S175P, T177K S173P, S175P, F180Y, L184H I137E, S173P, S175P, F180Y, V185K, T297P I137E, S173P, S175P, F180Y, V185K, N186P, T297P I137E, S173P, S175P, F180Y, V185K, N186D, T297P I137E, S173P, S175P, F180Y, N186K, T297P S173P, S175P, N186K I137E, S173P, S175P, F180Y, N186R, T297P S173P, S175P, E194* S173P, S175P, E194*, Q198E S173P, S175P, F180Y, E194*, Q198E, T297P S173P, S175P, F180Y, E194S S27K, E127Q, S173P, S175P, F180Y, E194S, Q198E, T297P S173P, S175P, F180Y, E194K S27K, E127Q, S173P, S175P, F180Y, E194K, Q198E, T297P S27K, E127Q, S173P, S175P, F180Y, E194Q, Q198E, T297P S173P, S175P, N195H S27K, E127Q, S173P, S175P, F180Y, N195H, V196N, Q197*, Q198*, N199*, G200*, T201*, T297P S173P, S175P, N195K S173P, S175P, V196H S173P, S175P, Q197K S27K, S173P, S175P, F180Y, Q197K, T297P S27K, S173P, S175P, F180Y, E194R, Q197K, Q198E, T297P S27K, S173P, S175P, F180Y, Q197K, Q198E, T297P S27K, E127Q, S173P, S175P, F180Y, Q197K, Q198E, T297P S27K, S171N, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P S27K, N109S, S111R, S173P, S175P, F180Y, Q197K, Q198E, T297P S27K, S173P, G174K, S175P, F180Y, L184F, Q197K, Q198E, T297P I121V, S173P, S175P, F180Y, Q197K, Q198E, T297P S173P, S175P, Q197H S173P, S175P, F180Y, Q197R S27K, S171N, S173P, G174R, S175P, F180Y, Q197R, Q198E, T297P S173P, S175P, Q198K S173P, S175P, Q198K S173P, S175P, N199K S173P, S175P, N199K S27K, S173P, S175P, F180Y, E194*, Q197K, Q198E, N199K, T297P S27K, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P S27K, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P S27K, I121V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, N109S, S111R, S173P, S175P, F180Y, Q198E, N199K, T297P S27K, S173P, S175P, F180Y, Q197K, Q198E, N199K, T297P S27K, Q70N, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, S114V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, Q70N, G107N, I121V, E127Q, S173P, S175P, F180Y, Q198E, N199K, T297P S27K, Q70A, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, Q70V, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, S171R, S173P, G174E, S175P, F180Y, Q198E, N199K, T297P S27K, S171K, S173P, G174E, S175P, F180Y, Q198E, N199K, T297P S27K, S171E, S173P, G174K, S175P, F180Y, Q198E, N199K, T297P S173P, S175P, G200H S173P, S175P, D206N S173P, S175P, N212K S173P, S175P, F180Y, A214R S173P, S175P, F180Y, A214K S173P, S175P, T215K S173P, S175P, D218N S173P, S175P, D218H S173P, S175P, F180Y, D218R S173P, S175P, Y219R S173P, S175P, Y219H S173P, S175P, I220K S173P, S175P, Q222K S27K, Q97K, E127Q, S173P, S175P, F180Y, Q198E, D225N, I247M, T297P S173P, S175P, F180Y, A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y, S234K T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P A1*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P A1*, Q6C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P A1*, S4C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P A1*, P3*, T5C, S27K, S171N, S173P, G174R, S175P, F180Y, Q198E, E236C, T297P S173P, S175P, T241K S173P, S175P, N245K S173P, S175P, T246K S173P, S175P, S248K S173P, S175P, F180Y, I264R S173P, S175P, F180Y, I264R S173P, S175P, F180Y, I264R S173P, S175P, F180Y, I264R S173P, S175P, A267H S173P, S175P, N268K S27K, E127Q, S173P, S175P, F180Y, Q198E, N268K, T297P S173P, S175P, T269K S173P, S175P, S270K S173P, S175P, S270K S173P, S175P, F180Y, Q198E, S270K, T297P S27K, S173P, S175P, F180Y, Q198E, S270K, T297P S27K, I137E, S173P, S175P, F180Y, S270K, T297P S27K, N87K, I121V, S171N, S173P, S175P, F180Y, S270K, T297P S27K, S173P, S175P, F180Y, S270K, T297P S27K, S173P, S175P, F180Y, S270K, T297P S173P, S175P, S272K S173P, S175P, S274K S173P, S175P, T278K S173P, S175P, E279Q S173P, S175P, E279H S173P, S175P, V286H I137E, S173P, S175P, F180Y, V286K, T297P I137E, S173P, S175P, F180Y, V286K, T297P S173P, S175P, Y287R S173P, S175P, Y287H I137E, S173P, S175P, F180Y, D288N, T297P S173P, S175P, F180Y, Q198E, D288N, T297P I137E, S173P, S175P, F180Y, D288K, T297P S27K, S173P, S175P, F180Y, Q198E, I293R, T297P S27K, I137E, S173P, S175P, F180Y, I293R, T297P S173P, S175P, I293K S173P, S175P, T297K G28D, S86K, N87R, Q89R, S173P, S175P, K311R G28N, S86K, N87R, Q89R, S173P, S175P, K311R b - E127S, S173P, S175P, F180Y S173P, S175P, F180Y S173P, S175P, F180Y, D299R S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q197K, Q198E, S274V, T297P S27K, D147N, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, D147N, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P S27K, D147S, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, G142E, D147N, I150S, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, G142E, I150S, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, T297P S27K, I150L, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, I150S, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, I150S, S171N, S173P, G174R, S175P, F180Y, Q198E, N199K, R224G, T297P S27K, N109K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, N109K, S111D, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, N109K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, N109K, S111E, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, N109K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, N109S, *109aE, S111R, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, N109S, *109aE, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, N109S, I121V, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, N87K, G132H, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, N87K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P S27K, N87K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, N87K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, N87K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P S27K, N87K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, Q70A, D147N, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, Q70A, N109K, S111E, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, Q70A, S111E, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, Q70A, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S27K, S111D, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, S111D, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, S111E, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, S111E, S171E, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P S27K, S111R, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P S27K, S114V, I121V, S129K, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P S27K, S114V, I121V, S173P, G174K, S175P, F180Y, L184F, Q198E, N199K, T297P S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S270V, S274V, L280V, T297P S27K, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, T297P S27K, S171D, S173P, G174R, S175P, F180Y, Q197K, Q198E, T297P S27K, S171D, S173P, G174R, S175P, F180Y, Q198E, T297P S27K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S27K, S173P, G174K, S175P, F180Y, G182A, L184F, Q198E, V286R, T297P S27K, V162T, S171D, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P S4K, S27K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P S4K, S27K, N87K, E127Q, G132H, Y156R, S173P, S175P, F180Y, Q198E, T297P T24R, S173P, S175P, F180Y

Example 2: Wash Testing of Variants by Automatic Mechanical Stress Assay (AMSA)

(241) Washing experiments are performed in order to assess the wash performance of selected protease variants in laundry detergent. The proteases of the present application were tested using the Automatic Mechanical Stress Assay (AMSA). With the AMSA, the wash performance of many small volume enzyme-detergent solutions can be examined. The AMSA plate has a number of slots for test solutions and a lid that firmly squeezes the textile swatch to be washed against the slot openings. During the wash, the plate, test solutions, textile swatch and lid are vigorously shaken to bring the test solution in contact with the soiled textile swatch and apply mechanical stress in a regular, periodic oscillating manner. For further description see WO 02/42740 especially the paragraph “Special method embodiments” on page 23-24.

(242) The experiment was conducted under the experimental conditions as specified in Table 3 below.

(243) TABLE-US-00003 TABLE 3 AMSA Experimental Conditions Detergent Model detergent B Detergent dosage 3.33 g/L Test solution volume 160 μL Wash time 20 minutes Temperature 20° C. Water hardness 15° dH Enzyme concentration in test solution 30-60 nM Test material Chocolate milk and soot swatch (PC-03) Blood Milk Ink swatch (EMPA117) with extra heat treatment, Full egg with pigment swatch (CS-37)
Water hardness was adjusted to 15° dH by addition of CaCl2, MgCl2, and NaHCO3 (Ca2+:Mg2+:CO32−=4:1:7.5) to the test system. The swatch textiles were flushed in tap water and dried after washing.

(244) The performance of an enzyme variant is measured as the brightness of the colour of the textile washed with that specific protease. Brightness can also be expressed as the intensity of the light reflected from the sample when illuminated with white light. When the sample is stained the intensity of the reflected light is lower, than that of a clean sample. Therefore the intensity of the reflected light can be used to measure wash performance of a protease. Color measurements were made with a professional flatbed scanner (EPSON EXPRESSION 10000XL, Atea NS, Lautrupvang 6, 2750 Ballerup, Denmark), which is used to capture an image of the washed melamine tiles.

(245) To extract a value for the light intensity from the scanned images, a special designed software application is used (Novozymes Colour Vector Analyzer). The program retrieves the 24 bit pixel values from the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
Int=√{square root over (r.sup.2+g.sup.2+b.sup.2)}
Textiles

(246) Standard chocolate milk and soot textile swatches (PC-03), blood milk ink (EMPA117) with extra heat treatment and full egg with pigment swatch (CS-37) textile swatches were obtained from Center For Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen, the Netherlands.

(247) The AMSA relative wash performance is then calculated for each variant by dividing the intensity value for textile washed with the variant by the intensity value for the textile washed with the parent backbone

(248) $\mathrm{AMSA}\mathrm{relative}\mathrm{wash}{\mathrm{performance}}_{\mathrm{variant}}=\frac{{\mathrm{Int}}_{\mathrm{variant}}}{{\mathrm{Int}}_{\mathrm{parent}}}$

(249) Having the AMSA relative wash performance for each variant, it is possible to calculate the effect of a single mutation, if data are available for at least two variants that differ only by that mutation (Table 4). In this case, the AMSA relative wash performance for the mutation is calculated:

(250) $\mathrm{AMSA}\mathrm{relative}\mathrm{wash}{\mathrm{performance}}_{\mathrm{mutation}}=\frac{\mathrm{AMSA}\mathrm{relative}\mathrm{wash}{\mathrm{performance}}_{\mathrm{variant}\mathrm{with}\mathrm{mutation}}}{\mathrm{AMSA}\mathrm{relative}\mathrm{wash}{\mathrm{performance}}_{\mathrm{variant}\mathrm{without}\mathrm{mutation}}}$

(251) TABLE-US-00004 TABLE 4 AMSA relative wash performance.sub.mutation on standard chocolate milk and soot textile swatch (PC-03) and a standard blood milk ink with extra heat treatment textile swatch (EMPA117EH) Mutation Variant with the mutation Variant without the mutation PC-03 EMPA117EH a: V2H V2H, S4K, S27K, S173P, S175P, S4K, S27K, S173P, S175P, F180Y, 1.16 0.99 F180Y, Q198E, T297P Q198E, T297P S4K S4K, S173P, S175P S173P, S175P 1.47 1.05 S4K S4K, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.76 1.23 T297P T297P S4K S4K, S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, Q198E, 1.22 1.08 Q198E, T297P T297P S4K S4K, S27K, I137E, S173P, S175P, S27K, I137E, S173P, S175P, F180Y, 1.37 1.10 F180Y, T297P T297P S4K A1*, V2*, P3*, S4K, S27K, S173P, A1*, V2*, P3*, S27K, S173P, S175P, 1.63 1.27 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P S4K S4K, S27K, N87K, E127Q, G132H, S27K, N87K, E127Q, G132H, S173P, 1.38 1.17 S173P, S175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P S4H S4H, S173P, S175P S173P, S175P 1.30 1.05 T5K T5K, S173P, S175P S173P, S175P 1.49 1.15 Q6K Q6K, S173P, S175P S173P, S175P 1.33 1.03 N16K N16K, S173P, S175P S173P, S175P 1.54 1.05 N16K N16K, D17N, S27K, S173P, S175P, D17N, S27K, S173P, S175P, F180Y, 1.23 1.11 F180Y, Q198E, T297P Q198E, T297P N16H N16H, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.12 1.13 T297P T297P N16H N16H, S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, Q198E, 0.84 1.06 Q198E, T297P T297P N16H N16H, S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, 1.21 1.10 Q198E, T297P Q198E, T297P N16H N16H, S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, 1.13 — Q198E, T297P Q198E, T297P N16H N16H, S27K, I137E, S173P, S175P, S27K, I137E, S173P, S175P, F180Y, 1.22 1.11 F180Y, T297P T297P D17N D17N, S173P, S175P S173P, S175P 1.42 1.10 D17N D17N, I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, 0.93 1.19 T297P T297P D17N D17N, S173P, S175P, Q198E S173P, S175P, Q198E 1.06 1.22 D17N D17N, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.43 1.18 T297P T297P D17N D17N, S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, Q198E, 1.28 0.99 Q198E, T297P T297P D17N D17N, S27K, I137E, S173P, S175P, S27K, I137E, S173P, S175P, F180Y, 1.37 1.05 F180Y, T297P T297P Q18K Q18K, S173P, S175P S173P, S175P 1.40 1.10 Q18K Q18K, S27K, E127Q, S173P, S175P, S27K, E127Q, S173P, S175P, F180Y, 1.26 0.97 F180Y, Q198E, T297P Q198E, T297P S19K S19K, S173P, S175P S173P, S175P 1.29 1.02 T24K T24K, S173P, S175P S173P, S175P 1.52 1.04 T24H T24H, S173P, S175P S173P, S175P 1.24 0.94 S27K S27K, S173P, S175P S173P, S175P 1.19 1.04 S27K S27K, I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, 0.84 1.09 T297P T297P S27K S27K, S173P, S175P, Q198E S173P, S175P, Q198E 0.93 1.16 S27K S27K, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.67 1.29 T297P T297P S27K S27K, S144R, S173P, S175P, F180Y, S144R, S173P, S175P, F180Y, 1.24 1.12 Q198E, T297P Q198E, T297P S27K S27K, D147N, S173P, S175P, F180Y, D147N, S173P, S175P, F180Y, 1.17 1.05 Q198E, T297P Q198E, T297P S27K S27K, E127Q, S173P, S175P, F180Y, E127Q, S173P, S175P, F180Y, 1.18 1.09 Q198E, T297P Q198E, T297P S27K S27K, E50Q, S173P, S175P, F180Y, E50Q, S173P, S175P, F180Y, 1.37 1.12 Q198E, T297P Q198E, T297P S27K S27K, N87K, S173P, S175P, F180Y, N87K, S173P, S175P, F180Y, 1.40 1.10 Q198E, T297P Q198E, T297P S27K S4K, S27K, S173P, S175P, F180Y, S4K, S173P, S175P, F180Y, Q198E, 1.16 1.13 Q198E, T297P T297P S27K S27K, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.44 1.12 S270K, T297P S270K, T297P S27K S27K, Q97K, S173P, S175P, F180Y, Q97K, S173P, S175P, F180Y, Q198E, 1.27 1.03 Q198E, T297P T297P S27K D17N, S27K, S173P, S175P, F180Y, D17N, S173P, S175P, F180Y, Q198E, 1.49 1.09 Q198E, T297P T297P S27K N16H, S27K, S173P, S175P, F180Y, N16H, S173P, S175P, F180Y, Q198E, 1.25 1.21 Q198E, T297P T297P S27K N16H, S27K, S173P, S175P, F180Y, N16H, S173P, S175P, F180Y, Q198E, 1.80 1.26 Q198E, T297P T297P S27K N16H, S27K, S173P, S175P, F180Y, N16H, S173P, S175P, F180Y, Q198E, 1.68 — Q198E, T297P T297P S27K S27K, I137E, S144R, S173P, S175P, I137E, S144R, S173P, S175P, F180Y 1.54 1.04 F180Y, T297P T297P S27K S27K, E127Q, I137E, S173P, S175P, E127Q, I137E, S173P, S175P, F180Y, 1.20 1.05 F180Y, T297P T297P S27K S27K, I137E, S173P, G174R, S175P, I137E, S173P, G174R, S175P, F180Y, 1.14 1.09 F180Y, T297P T297P S27K S27K, Q97K, I137E, S173P, S175P, Q97K, I137E, S173P, S175P, F180Y, 1.52 1.03 F180Y, T297P T297P S27K D17N, S27K, I137E, S173P, S175P, D17N, I137E, S173P, S175P, F180Y, 1.24 0.96 F180Y, T297P T297P S27K S27K, D126G, S173P, S175P, F180Y, D126G, S173P, S175P, F180Y, 1.05 1.06 T297P T297P Y39R Y39R, S173P, S175P S173P, S175P 1.10 0.98 Y39R S27K, Y39R, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, Q198E, 1.31 1.03 Q198E, T297P T297P Y39R S27K, Y39R, I137E, S173P, S175P, S27K, I137E, S173P, S175P, F180Y, 1.28 1.14 F180Y, T297P T297P Y39R S27K, Y39R, N87K, I137E, S173P, S27K, N87K, I137E, S173P, S175P, 1.10 1.07 S175P, F180Y, T297P F180Y, T297P Y39R S27K, Y39R, I137E, S144R, S173P, S27K, I137E, S144R, S173P, S175P, 1.21 0.95 S175P, F180Y, T297P F180Y, T297P Y39R S27K, Y39R, I121V, S171N, S173P, S27K, I121V, S171N, S173P, G174R, 1.11 0.88 G174R, S175P, F180Y, Q198E, S175P, F180Y, Q198E, T297P T297P S41K S41K, S173P, S175P S173P, S175P 1.48 1.16 S41K S27K, S41K, E127Q, S173P, S175P, S27K, E127Q, S173P, S175P, F180Y, 1.11 1.01 F180Y, Q198E, T297P Q198E, T297P L43H L43H, S173P, S175P S173P, S175P 1.16 1.02 G47K G47K, R130K, G132R, S173P, R130K, G132R, S173P, S175P 1.59 1.26 S175P E50Q E50Q, S173P, S175P S173P, S175P 1.26 1.10 E50Q E50Q, S173P, S175P, F180Y S173P, S175P, F180Y 1.77 1.25 E50Q E50Q, S173P, S175P, Q198E S173P, S175P, Q198E 0.94 1.10 E50Q E50Q, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.55 1.20 Q198E, T297P T297P E50Q S27K, E50Q, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, Q198E, 1.27 1.04 Q198E, T297P T297P E50T E50T, S173P, S175P, F180Y S173P, S175P, F180Y 1.08 1.16 E50L E50L, S173P, S175P, F180Y S173P, S175P, F180Y 1.83 1.27 Q57K G28N, Q57K, S86K, N87R, Q89R, G28N, S86K, N87R, Q89R, S173P, 1.05 0.96 S173P, S175P S175P S58K S58K, S173P, S175P S173P, S175P 1.27 0.98 S58K S58K, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.57 1.22 Q198E, T297P T297P N59K N59K, S173P, S175P S173P, S175P 1.18 1.04 D63N D63N, S173P, S175P S173P, S175P 1.12 1.06 D63E D63E, R69D, S173P, S175P R69D, S173P, S175P 1.41 1.03 G64H G64H, S173P, S175P S173P, S175P 1.19 0.96 S65K S65K, S173P, S175P S173P, S175P 1.22 0.96 S65K S65K, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.63 1.20 Q198E, T297P T297P S65K S27K, S65K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, T297P 1.07 0.94 T297P S65K S27K, S65K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, T297P 1.13 — T297P T67K T67K, S173P, S175P S173P, S175P 1.12 0.98 Q70H Q70H, S173P, S175P, F180Y S173P, S175P, F180Y 1.09 1.12 G85R G85R, S173P, S175P, F180Y S173P, S175P, F180Y 1.84 1.26 G85H G85H, S173P, S175P, F180Y S173P, S175P, F180Y 2.17 1.34 S86K S86K, S173P, S175P S173P, S175P 1.10 1.01 N87K N87K, S173P, S175P S173P, S175P 1.44 1.11 N87K N87K, I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, 0.98 1.12 T297P T297P N87K N87K, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.47 1.22 Q198E, T297P T297P N87K S27K, N87K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, 1.24 1.03 Q198E, T297P Q198E, T297P N87K S27K, N87K, I137E, S173P, S175P, S27K, I137E, S173P, S175P, 1.11 0.96 F180Y, T297P F180Y, T297P N87K S27K, N87K, N109S, S111R, S173P, S27K, N109S, S111R, S173P, 1.15 1.03 S175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P N87K S27K, N59L, T67V, N87K, G107N, S27K, N59L, T67V, G107N, I121V, 1.06 1.08 I121V, S171N, S173P, G174R, S175P, S171N, S173P, G174R, S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P N87K S27K, N59L, T67V, N87K, G107N, S27K, N59L, T67V, G107N, I121V, 1.08 1.08 I121V, E127Q, S171N, S173P, E127Q, S171N, S173P, G174R, S175P, G174R, S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P N87K S27K, Q70N, N87K, I121V, E127Q, S27K, Q70N, I121V, E127Q, S171N, 1.09 0.95 S171N, S173P, G174R, S175P, S173P, G174R, S175P, F180Y, F180Y, Q198E, N199R, T297P Q198E, N199R, T297P Y92R Y92R, S173P, S175P, F180Y S173P, S175P, F180Y 1.73 1.28 Q97K Q97K, S173P, S175P S173P, S175P 1.18 1.06 Q97K Q97K, I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, T297P 0.81 1.12 T297P Q97K Q97K, S173P, S175P, Q198E S173P, S175P, Q198E 0.89 1.10 Q97K Q97K, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.49 1.21 Q198E, T297P T297P Q97K S27K, Q97K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, 1.13 0.97 Q198E, T297P Q198E, T297P Q97K S27K, Q97K, I137E, S173P, S175P, S27K, I137E, S173P, S175P, F180Y, 1.47 1.06 F180Y, T297P T297P Q97K S27K, N59L, T67V, Q97K, G107N, S27K, N59L, T67V, G107N, I121V, 1.05 1.01 I121V, S171N, S173P, G174R, S171N, S173P, G174R, S175P, S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P D108N D108N, S173P, S175P S173P, S175P — 1.10 D108S D108S, S173P, S175P S173P, S175P 1.11 0.99 D108A D108A, S173P, S175P S173P, S175P 1.19 1.00 N109K S27K, N109K, S173P, S175P, S27K, S173P, S175P, F180Y, 1.26 1.04 F180Y, Q198E, T297P Q198E, T297P S111K S111K, S173P, S175P S173P, S175P 1.19 0.93 S111R S27K, N109S, S111R, S173P, S27K, N109S, S173P, S175P, F180Y, 1.61 1.09 S175P, F180Y, Q198E, T297P Q198E, T297P S114K S114K, S173P, S175P, F180Y S173P, S175P, F180Y 1.61 1.11 D116S D116S, M139L, S173P, S175P M139L, S173P, S175P 2.70 1.38 A118R A118R, S173P, S175P, F180Y S173P, S175P, F180Y 1.61 1.20 A118R S27K, S111R, A118R, S173P, S27K, S111R, S173P, S175P, 1.76 0.94 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P A118K A118K, S173P, S175P, F180Y S173P, S175P, F180Y 1.60 1.20 D126G D126G, S173P, S175P, F180Y S173P, S175P, F180Y 1.95 1.24 D126G S27K, D126G, S173P, S175P, S27K, S173P, S175P, F180Y, 1.15 0.99 F180Y, Q198E, T297P Q198E, T297P D126G S27K, D126G, E127Q, S173P, S27K, E127Q, S173P, S175P, 1.10 0.87 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P D126W D126W, S173P, S175P, F180Y S173P, S175P, F180Y 1.82 1.20 D126K D126K, S173P, S175P, F180Y S173P, S175P, F180Y 1.78 1.18 E127Q Y113T, E127Q, S173P, G174K, Y113T, S173P, G174K, S175P, 1.13 0.99 S175P, F180Y F180Y E127Q *109aS, E127Q, S173P, S175P, *109aS, S173P, S175P, F180Y, 2.38 1.71 F180Y, T297P T297P E127Q E127Q, S173P, S175P, Q198E S173P, S175P, Q198E 1.06 1.21 E127Q E127Q, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.67 1.23 Q198E, T297P T297P E127Q S27K, E127Q, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, Q198E, 1.19 1.04 Q198E, T297P T297P E127Q S27K, D126G, E127Q, S173P, S175P, S27K, D126G, S173P, S175P, F180Y, 1.14 0.91 F180Y, Q198E, T297P Q198E, T297P E127Q S27K, E127Q, I137Q, S173P, S175P, S27K, I137Q, S173P, S175P, F180Y, 1.70 1.14 F180Y, Q198E, T297P Q198E, T297P E127Q S27K, E127Q, S144R, S173P, S27K, S144R, S173P, S175P, 1.14 0.97 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162S, S173P, S27K, V162S, S173P, S175P, 1.89 1.19 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162E, S173P, S27K, V162E, S173P, S175P, 1.44 1.07 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162Q, S173P, S27K, V162Q, S173P, S175P, 1.23 1.04 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162G, S173P, S27K, V162G, S173P, S175P, 1.34 1.07 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162F, S173P, S27K, V162F, S173P, S175P, 1.36 1.07 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162T, S173P, S175P, S27K, V162T, S173P, S175P, 1.99 1.25 F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, E127Q, V162Y, S173P, S27K, V162Y, S173P, S175P, 1.72 1.11 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, N109S, S111R, E127Q, S27K, N109S, S111R, S173P, 1.25 1.18 S173P, 175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P E127Q S27K, N109S, S111R, E127Q, S27K, N109S, S111R, S173P, 1.44 1.21 S173P, 175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P E127Q S27K, N59L, T67V, N87K, G107N, S27K, N59L, T67V, N87K, G107N, 1.06 0.98 I121V, E127Q, S171N, S173P, G174R, I121V, S171N, S173P, G174R, S175P, S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P E127Q S27K, N87K, I121V, E127Q, S171N, S27K, N87K, I121V, S171N, S173P, 1.06 0.99 S173P, S175P, F180Y, T297P S175P, F180Y, T297P E127K E127K, S173P, S175P, F180Y S173P, S175P, F180Y 1.72 1.16 E127L E127L, S173P, S175P, F180Y S173P, S175P, F180Y 1.27 1.30 S129K S129K, S173P, S175P S173P, S175P 1.25 1.16 R130K S41K, L43K, G47K, R130K, G132R, S41K, L43K, G47K, G132R, S173P, 1.21 1.02 S173P, S175P S175P R130K S41R, L43R, G47K, R130K, G132R, S41R, L43R, G47K, G132R, S173P, 1.11 0.98 S173P, S175P S175P T131K T131K, S173P, S175P S173P, S175P 1.44 1.03 G132H G132H, S173P, S175P S173P, S175P 1.18 1.02 G132H G132H, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.11 1.11 Q198E, T297P T297P S143K S143K, S173P, S175P S173P, S175P 1.10 0.96 S144K S144K, S173P, S175P S173P, S175P 1.32 0.99 D147N D147N, S173P, S175P S173P, S175P 1.51 0.89 D147N D147N, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.55 1.09 T297P T297P D147N S27K, D147N, S173P, S175P, S27K, S173P, S175P, F180Y, 1.09 0.89 F180Y, Q198E, T297P Q198E, T297P D147N S27K, N109S, S111R, D147N, S173P, S27K, N109S, S111R, S173P, S175P, 1.11 1.00 S175P, F180Y, Q198E, T297P F180Y, Q198E, T297P D147S D147S, S148A, L149T, I150L, S148A, L149T, I150L, S173P, S175P 1.69 1.13 S173P, S175P D147S A145S, K146P, D147S, S173P, A145S, K146P, S173P, S175P 1.74 1.01 S175P D147S A145S, K146P, D147S, S148A, A145S, K146P, S148A, S173P, 2.04 1.09 S173P, S175P S175P D147S S144P, A145S, K146P, D147S, S144P, A145S, K146P, S148A, 2.41 1.20 S148A, L149T, I150L, S173P, S175P L149T, I150L, S173P, S175P D147S D147S, L149T, S173P, S175P L149T, S173P, S175P 1.41 1.08 D147S K146P, D147S, S148A, L149T, K146P, S148A, L149T, I150L, S173P, 1.33 1.14 I150L, S173P, S175P S175P D147S A145S, K146P, D147S, S148A, A145S, K146P, S148A, L149T, 1.18 1.08 L149T, S173P, S175P S173P, S175P D147S S27K, K146E, D147S, S171N, S27K, K146E, S171N, S173P, G174R, 1.16 0.83 S173P, G174R, S175P, F180Y, S175P, F180Y, G182A, Q198E, G182A, Q198E, N199K, E223K, T297P N199K, E223K, T297P D147* K146*, D147*, S173P, S175P K146*, S173P, S175P — 1.08 D147Q D147Q, S173P, S175P S173P, S175P 1.28 0.84 D147Q D147Q, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.40 1.00 Q198E, T297P T297P D147H D147H, S173P, S175P S173P, S175P 1.31 0.85 S148K S148K, S173P, S175P S173P, S175P 1.33 1.00 L149K L149K, S173P, S175P S173P, S175P 1.12 0.99 D155H D155H, S173P, S175P S173P, S175P 1.25 0.98 Y156R Y156R, S173P, S175P S173P, S175P 1.29 0.95 Y156R Y156R, S173P, S175P S173P, S175P 1.33 1.03 Y156R Y156R, S173P, S175P S173P, S175P 1.19 0.93 Y156R Y156R, S173P, S175P, Q198E S173P, S175P, Q198E 1.04 1.12 Y156R Y156R, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, 1.59 1.19 Q198E, T297P T297P Y156R S4K, D17N, S27K, N59L, N87K, S4K, D17N, S27K, N59L, N87K, 1.12 0.92 Y156R, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, Q198E, T297P T297P Y156R S4K, D17N, S27K, N59L, N87K, S4K, D17N, S27K, N59L, N87K, 1.18 — Y156R, S173P, S175P, F180Y, S173P, S175P, F180Y, Q198E, Q198E, T297P T297P Y156H Y156H, S173P, S175P S173P, S175P 1.18 0.97 G159H G159H, S173P, S175P S173P, S175P 1.21 1.05 G174R S27K, S173P, G174R, S175P, S27K, S173P, S175P, F180Y, 1.18 1.00 F180Y, Q198E, T297P Q198E, T297P G174R S27K, I137E, S173P, G174R, S175P, S27K, I137E, S173P, S175P, F180Y, 1.15 1.04 F180Y, T297P T297P G174R S27K, N59L, T67V, N87K, S114V, S27K, N59L, T67V, N87K, S114V, 1.07 0.91 I121V, I150L, S171N, S173P, G174R, I121V, I150L, S171N, S173P, S175P, S175P, F180Y, T297P F180Y, T297P T177K S173P, S175P, T177K S173P, S175P 1.09 0.93 L184H S173P, S175P, F180Y, L184H S173P, S175P, F180Y 1.32 1.21 V185K I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, T297P 1.00 1.13 V185K, T297P V185K I137E, S173P, S175P, F180Y, V185K, I137E, S173P, S175P, F180Y, 1.49 1.14 N186P, T297P N186P, T297P V185K I137E, S173P, S175P, F180Y, V185K, I137E, S173P, S175P, F180Y, 1.72 1.23 N186D, T297P N186D, T297P N186K I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, T297P 0.84 1.08 N186K, T297P N186K S173P, S175P, N186K S173P, S175P 1.22 1.04 N186R I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, T297P 0.83 1.13 N186R, T297P E194* S173P, S175P, E194* S173P, S175P 1.34 0.98 E194* S173P, S175P, E194*, Q198E S173P, S175P, Q198E 0.85 1.12 E194* S173P, S175P, F180Y, E194*, S173P, S175P, F180Y, Q198E, 1.54 1.26 Q198E, T297P T297P E194S S173P, S175P, F180Y, E194S S173P, S175P, F180Y 2.05 1.25 E194S S27K, E127Q, S173P, S175P, S27K, E127Q, S173P, S175P, 1.28 0.95 F180Y, E194S, Q198E, T297P F180Y, Q198E, T297P E194K S173P, S175P, F180Y, E194K S173P, S175P, F180Y 1.84 1.13 E194K S27K, E127Q, S173P, S175P, S27K, E127Q, S173P, S175P, 1.30 0.91 F180Y, E194K, Q198E, T297P F180Y, Q198E, T297P E194Q S27K, E127Q, S173P, S175P, S27K, E127Q, S173P, S175P, 1.27 0.96 F180Y, E194Q, Q198E, T297P F180Y, Q198E, T297P N195H S173P, S175P, N195H S173P, S175P 1.09 0.99 N195H S27K, E127Q, S173P, S175P, S27K, E127Q, S173P, S175P, 0.96 1.06 F180Y, N195H, V196N, Q197*, F180Y, V196N, Q197*, Q198*, Q198*, N199*, G200*, T201*, T297P N199*, G200*, T201*, T297P N195K S173P, S175P, N195K S173P, S175P 1.25 1.07 V196H S173P, S175P, V196H S173P, S175P 1.36 0.98 Q197K S173P, S175P, Q197K S173P, S175P 1.20 0.95 Q197K S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, T297P 1.07 1.05 Q197K, T297P Q197K S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, 1.10 0.96 E194R, Q197K, Q198E, T297P E194R, Q198E, T297P Q197K S27K, S173P, S175P, F180Y, S27K, S173P, S175P, F180Y, 1.38 1.14 Q197K, Q198E, T297P Q198E, T297P Q197K S27K, E127Q, S173P, S175P, F180Y, S27K, E127Q, S173P, S175P, F180Y, 1.17 1.01 Q197K, Q198E, T297P Q198E, T297P Q197K S27K, S171N, S173P, G174R, S175P, S27K, S171N, S173P, G174R, S175P, 1.17 1.11 F180Y, Q197K, Q198E, T297P F180Y, Q198E, T297P Q197K S27K, N109S, S111R, S173P, S175P, S27K, N109S, S111R, S173P, S175P, 1.12 0.92 F180Y, Q197K, Q198E, T297P F180Y, Q198E, T297P Q197K S27K, S173P, G174K, S175P, F180Y, S27K, S173P, G174K, S175P, F180Y, 1.57 1.03 L184F, Q197K, Q198E, T297P L184F, Q198E, T297P Q197K I121V, S173P, S175P, F180Y, I121V, S173P, S175P, F180Y, 1.50 1.30 Q197K, Q198E, T297P Q198E, T297P Q197H S173P, S175P, Q197H S173P, S175P 1.16 1.01 Q197R S173P, S175P, F180Y, Q197R S173P, S175P, F180Y 2.50 1.38 Q197R S27K, S171N, S173P, G174R, S175P, S27K, S171N, S173P, G174R, 1.05 — F180Y, Q197R, Q198E, T297P S175P, F180Y, Q198E, T297P Q198K S173P, S175P, Q198K S173P, S175P 1.39 1.00 Q198K S173P, S175P, Q198K S173P, S175P 1.36 0.99 N199K S173P, S175P, N199K S173P, S175P 1.34 0.99 N199K S173P, S175P, N199K S173P, S175P 1.35 1.03 N199K S27K, S173P, S175P, F180Y, E194*, S27K, S173P, S175P, F180Y, E194*, 1.18 0.96 Q197K, Q198E, N199K, T297P Q197K, Q198E, T297P N199K S27K, E127Q, S173P, S175P, F180Y, S27K, E127Q, S173P, S175P, F180Y, 1.27 1.04 Q198E, N199K, T297P Q198E, T297P N199K S27K, E127Q, S173P, S175P, F180Y, S27K, E127Q, S173P, S175P, F180Y, 1.37 — Q198E, N199K, T297P Q198E, T297P N199K S27K, I121V, S171N, S173P, G174R, S27K, I121V, S171N, S173P, G174R, 1.09 1.01 S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P N199K S27K, S171N, S173P, G174R, S27K, S171N, S173P, G174R, 1.23 1.05 S175P, F180Y, Q198E, N199K, T297P S175P, F180Y, Q198E, T297P N199K S27K, N109S, S111R, S173P, S175P, S27K, N109S, S111R, S173P, S175P, 1.07 1.06 F180Y, Q198E, N199K, T297P F180Y, Q198E, T297P N199K S27K, S173P, S175P, F180Y, Q197K, S27K, S173P, S175P, F180Y, Q197K, 1.17 0.94 Q198E, N199K, T297P Q198E, T297P N199K S27K, Q70N, S171N, S173P, G174R, S27K, Q70N, S171N, S173P, G174R, 1.16 0.99 S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P N199K S27K, I150S, S171N, S173P, G174R, S27K, I150S, S171N, S173P, G174R, 1.25 1.03 S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P N199K S27K, S114V, S171N, S173P, G174R, S27K, S114V, S171N, S173P, G174R, 1.12 1.04 S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P N199K S27K, Q70N, G107N, I121V, E127Q, S27K, Q70N, G107N, I121V, E127Q, 1.08 1.00 S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, N199K, T297P T297P N199K S27K, Q70A, S171N, S173P, G174R, S27K, Q70A, S171N, S173P, G174R, 1.00 1.09 S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P N199K S27K, Q70V, S171N, S173P, G174R, S27K, Q70V, S171N, S173P, G174R, 1.31 0.95 S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P N199K S27K, S173P, G174K, S175P, F180Y, S27K, S173P, G174K, S175P, F180Y, 1.90 1.02 L184F, Q198E, N199K, T297P L184F, Q198E, T297P N199K S27K, S171D, S173P, G174R, S175P, S27K, S171D, S173P, G174R, S175P, 1.11 1.16 F180Y, Q198E, N199K, T297P F180Y, Q198E, T297P N199K S27K, S171R, S173P, G174E, S175P, S27K, S171R, S173P, G174E, S175P, 1.02 1.13 F180Y, Q198E, N199K, T297P F180Y, Q198E, T297P N199K S27K, S171K, S173P, G174E, S175P, S27K, S171K, S173P, G174E, S175P, 1.21 1.17 F180Y, Q198E, N199K, T297P F180Y, Q198E, T297P N199K S27K, S171E, S173P, G174K, S175P, S27K, S171E, S173P, G174K, S175P, 1.31 1.14 F180Y, Q198E, N199K, T297P F180Y, Q198E, T297P G200H S173P, S175P, G200H S173P, S175P 1.36 1.10 D206N S173P, S175P, D206N S173P, S175P — 1.12 N212K S173P, S175P, N212K S173P, S175P 1.13 0.94 A214R S173P, S175P, F180Y, A214R S173P, S175P, F180Y 1.63 1.23 A214K S173P, S175P, F180Y, A214K S173P, S175P, F180Y 1.80 1.32 T215K S173P, S175P, T215K S173P, S175P 1.46 1.04 D218N S173P, S175P, D218N S173P, S175P 1.46 1.09 D218H S173P, S175P, D218H S173P, S175P 1.20 1.03 D218R S173P, S175P, F180Y, D218R S173P, S175P, F180Y 1.73 1.27 Y219R S173P, S175P, Y219R S173P, S175P 1.24 1.01 Y219H S173P, S175P, Y219H S173P, S175P 1.10 0.96 I220K S173P, S175P, I220K S173P, S175P 1.26 1.05 Q222K S173P, S175P, Q222K S173P, S175P 1.25 1.02 D225N S27K, Q97K, E127Q, S173P, S175P, S27K, Q97K, E127Q, S173P, S175P, 0.99 1.10 F180Y, Q198E, D225N, I247M, F180Y, Q198E, I247M, T297P T297P A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y 1.92 1.24 A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y 1.69 1.29 A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y 1.98 — A233R S173P, S175P, F180Y, A233R S173P, S175P, F180Y 2.03 — S234K S173P, S175P, F180Y, S234K S173P, S175P, F180Y 1.78 1.25 E236C T5C, S27K, S171N, S173P, G174R, T5C, S27K, S171N, S173P, G174R, 1.21 1.08 S175P, F180Y, Q198E, E236C, T297P S175P, F180Y, Q198E, T297P E236C Q6C, S27K, S171N, S173P, G174R, Q6C, S27K, S171N, S173P, G174R, 1.51 1.13 S175P, F180Y, Q198E, E236C, S175P, F180Y, Q198E, T297P T297P E236C S4C, S27K, S171N, S173P, G174R, S4C, S27K, S171N, S173P, G174R, 1.06 0.94 S175P, F180Y, Q198E, E236C, T297P S175P, F180Y, Q198E, T297P E236C A1*, T5C, S27K, S171N, S173P, A1*, T5C, S27K, S171N, S173P, 1.71 1.21 G174R, S175P, F180Y, Q198E, E236C, G174R, S175P, F180Y, Q198E, T297P T297P E236C A1*, Q6C, S27K, S171N, S173P, A1*, Q6C, S27K, S171N, S173P, 1.07 1.02 G174R, S175P, F180Y, Q198E, E236C, G174R, S175P, F180Y, Q198E, T297P T297P E236C A1*, S4C, S27K, S171N, S173P, A1*, S4C, S27K, S171N, S173P, 1.27 1.07 G174R, S175P, F180Y, Q198E, E236C, G174R, S175P, F180Y, Q198E, T297P T297P E236C A1*, P3*, T5C, S27K, S171N, S173P, A1*, P3*, T5C, S27K, S171N, S173P, 1.34 1.06 G174R, S175P, F180Y, Q198E, G174R, S175P, F180Y, Q198E, E236C, T297P T297P T241K S173P, S175P, T241K S173P, S175P 1.34 0.99 N245K S173P, S175P, N245K S173P, S175P 1.38 1.03 T246K S173P, S175P, T246K S173P, S175P 1.64 1.05 S248K S173P, S175P, S248K S173P, S175P 1.07 0.96 I264R S173P, S175P, F180Y, I264R S173P, S175P, F180Y 1.81 1.36 I264R S173P, S175P, F180Y, I264R S173P, S175P, F180Y 1.66 1.37 I264R S173P, S175P, F180Y, I264R S173P, S175P, F180Y 1.94 — I264R S173P, S175P, F180Y, I264R S173P, S175P, F180Y 1.83 — A267H S173P, S175P, A267H S173P, S175P 1.10 0.95 N268K S173P, S175P, N268K S173P, S175P 1.39 1.05 N268K S27K, E127Q, S173P, S175P, F180Y, S27K, E127Q, S173P, S175P, F180Y, 1.18 0.92 Q198E, N268K, T297P Q198E, T297P T269K S173P, S175P, T269K S173P, S175P 1.29 1.05 S270K S173P, S175P, S270K S173P, S175P 1.27 1.08 S270K S173P, S175P, S270K S173P, S175P 1.25 1.07 S270K S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.40 1.22 S270K, T297P T297P S270K S27K, S173P, S175P, F180Y, Q198E, S27K, S173P, S175P, F180Y, Q198E, 1.21 1.06 S270K, T297P T297P S270K S27K, I137E, S173P, S175P, F180Y, S27K, I137E, S173P, S175P, F180Y, 1.19 1.02 S270K, T297P T297P S270K S27K, N87K, I121V, S171N, S173P, S27K, N87K, I121V, S171N, S173P, 1.11 1.01 S175P, F180Y, S270K, T297P S175P, F180Y, T297P S270K S27K, S173P, S175P, F180Y, S270K, S27K, S173P, S175P, F180Y, T297P 1.11 0.97 T297P S270K S27K, S173P, S175P, F180Y, S270K, S27K, S173P, S175P, F180Y, T297P 1.20 — T297P S272K S173P, S175P, S272K S173P, S175P 1.31 1.03 S274K S173P, S175P, S274K S173P, S175P 1.20 1.04 T278K S173P, S175P, T278K S173P, S175P 1.39 1.04 E279Q S173P, S175P, E279Q S173P, S175P 1.67 1.01 E279H S173P, S175P, E279H S173P, S175P 1.23 1.04 V286H S173P, S175P, V286H S173P, S175P 1.06 1.02 V286K I137E, S173P, S175P, F180Y, V286K, I137E, S173P, S175P, F180Y, T297P 0.93 1.14 T297P V286K I137E, S173P, S175P, F180Y, V286K, I137E, S173P, S175P, F180Y, T297P 0.73 1.07 T297P Y287R S173P, S175P, Y287R S173P, S175P 1.36 1.07 Y287H S173P, S175P, Y287H S173P, S175P 1.15 0.98 D288N I137E, S173P, S175P, F180Y, I137E, S173P, S175P, F180Y, T297P 0.77 1.09 D288N, T297P D288N S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, 1.34 1.19 D288N, T297P T297P D288K I137E, S173P, S175P, F180Y, D288K, I137E, S173P, S175P, F180Y, T297P 0.69 1.12 T297P I293R S27K, S173P, S175P, F180Y, Q198E, S27K, S173P, S175P, F180Y, Q198E, 1.17 1.03 I293R, T297P T297P I293R S27K, I137E, S173P, S175P, F180Y, S27K, I137E, S173P, S175P, F180Y, 1.11 1.03 I293R, T297P T297P I293K S173P, S175P, I293K S173P, S175P 1.36 1.05 T297K S173P, S175P, T297K S173P, S175P 1.42 1.13 K311R G28D, S86K, N87R, Q89R, S173P, G28D, S86K, N87R, Q89R, S173P, 1.51 1.13 S175P, K311R S175P K311R G28N, S86K, N87R, Q89R, S173P, G28N, S86K, N87R, Q89R, S173P, 0.95 1.06 S175P, K311R S175P b: D299R S173P, S175P, F180Y, D299R S173P, S175P, F180Y 1.38 1.11 N199K S27K, N109S, I121V, S171D, S173P, S27K, N109S, I121V, S171D, S173P, 1.39 1.17 G174R, S175P, F180Y, G182A, L184F, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P Q198E, S274V, T297P N199K S27K, I150S, S171D, S173P, G174R, S27K, I150S, S171D, S173P, G174R, 1.42 1.12 S175P, F180Y, G182A, L184F, Q198E, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S274V, T297P N199K S27K, Q70A, S171D, S173P, G174R, S27K, Q70A, S171D, S173P, G174R, 1.43 1.09 S175P, F180Y, G182A, L184F, Q198E, S175P, F180Y, G182A, L184F, Q198E, N199K, S274V, T297P S274V, T297P N199K S27K, I150L, S171N, S173P, G174R, S27K, I150L, S171N, S173P, G174R, 1.20 NA S175P, F180Y, Q198E, N199K, S175P, F180Y, Q198E, T297P T297P Q197K S27K, D147N, I150S, S171D, S173P, S27K, D147N, I150S, S171D, S173P, 1.25 1.04 G174R, S175P, F180Y, G182A, G174R, S175P, F180Y, G182A, L184F, L184F, Q197K, Q198E, S274V, T297P Q198E, S274V, T297P Q197K S27K, S171D, S173P, G174R, S175P, S27K, S171D, S173P, G174R, 0.98 1.09 F180Y, Q197K, Q198E, T297P S175P, F180Y, Q198E, T297P Q197K S27K, S171D, S173P, G174R, S175P, S27K, S171D, S173P, G174R, 1.16 1.14 F180Y, Q197K, Q198E, T297P S175P, F180Y, Q198E, T297P D147S S27K, D147S, I150S, S171D, S173P, S27K, I150S, S171D, S173P, G174R, 1.58 0.96 G174R, S175P, F180Y, G182A, L184F, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P Q198E, S274V, T297P D147N S27K, Q70A, D147N, S171D, S173P, S27K, Q70A, S171D, S173P, G174R, 1.20 0.94 G174R, S175P, F180Y, G182A, S175P, F180Y, G182A, L184F, L184F, Q198E, N199K, S274V, T297P Q198E, N199K, S274V, T297P D147N S27K, D147N, I150S, S171N, S173P, S27K, I150S, S171N, S173P, G174R, 1.13 0.96 G174R, S175P, F180Y, Q198E, S175P, F180Y, Q198E, N199K, N199K, R224G, T297P R224G, T297P D147N S27K, D147N, I150S, S171D, S173P, S27K, I150S, S171D, S173P, G174R, 1.67 1.01 G174R, S175P, F180Y, G182A, S175P, F180Y, G182A, L184F, L184F, Q198E, S274V, T297P Q198E, S274V, T297P D147N S27K, D147N, I150S, S171D, S173P, S27K, I150S, S171D, S173P, G174R, 1.39 1.03 G174R, S175P, F180Y, G182A, S175P, F180Y, G182A, L184F, L184F, Q198E, S274V, T297P Q198E, S274V, T297P G132H S27K, N87K, G132H, V162T, S171D, S27K, N87K, V162T, S171D, S173P, 1.15 1.04 S173P, G174R, S175P, F180Y, G182A, G174R, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P L184F, Q198E, S274V, T297P E127S E127S, S173P, S175P, F180Y S173P, S175P, F180Y 1.78 1.16 S111R S27K, N109S, *109aE, S111R, S171E, S27K, N109S, *109aE, S171E, S173P, 1.75 1.13 S173P, G174K, S175P, F180Y, G182A, G174K, S175P, F180Y, G182A, L184F, L184F, Q198E, N199K, T297P Q198E, N199K, T297P S111R S27K, S111R, S171D, S173P, G174R, S27K, S171D, S173P, G174R, S175P, 1.08 0.95 S175P, F180Y, G182A, L184F, Q198E, F180Y, G182A, L184F, Q198E, S270V, S270V, S274V, L280V, T297P S274V, L280V, T297P N109K S27K, Q70A, N109K, S111E, S171D, S27K, Q70A, S111E, S171D, S173P, 1.12 1.02 S173P, G174R, S175P, F180Y, G182A, G174R, S175P, F180Y, G182A, L184F, L184F, Q198E, N199K, S274V, Q198E, N199K, S274V, T297P T297P N109K S27K, N109K, S111E, S171E, S173P, S27K, S111E, S171E, S173P, G174K, 1.36 1.08 G174K, S175P, F180Y, G182A, S175P, F180Y, G182A, L184F, L184F, Q198E, N199K, T297P Q198E, N199K, T297P N109K S27K, N109K, S111D, S171E, S173P, S27K, S111D, S171E, S173P, G174K, 1.13 0.98 G174K, S175P, F180Y, G182A, S175P, F180Y, G182A, L184F, L184F, Q198E, N199K, T297P Q198E, N199K, T297P N109K S27K, N109K, S111E, S171E, S173P, S27K, S111E, S171E, S173P, G174K, 1.15 1.04 G174K, S175P, F180Y, G182A, S175P, F180Y, G182A, L184F, L184F, Q198E, N199K, T297P Q198E, N199K, T297P N109K S27K, N109K, S111D, S171D, S173P, S27K, S111D, S171D, S173P, G174R, 1.29 1.12 G174R, S175P, F180Y, Q198E, S175P, F180Y, Q198E, T297P T297P N109K S27K, N109K, S111E, S171D, S173P, S27K, S111E, S171D, S173P, G174R, 1.34 1.15 G174R, S175P, F180Y, Q198E, S175P, F180Y, Q198E, T297P T297P N109K S27K, N109K, S171D, S173P, G174R, S27K, S171D, S173P, G174R, 0.96 1.06 S175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P D147N S27K, G142E, D147N, I150S, S171N, S27K, G142E, I150S, S171N, S173P, 2.44 1.02 S173P, G174R, S175P, F180Y, G174R, S175P, F180Y, G182A, L184F, G182A, L184F, Q198E, N199K, T297P Q198E, N199K, T297P N87K S27K, N87K, S173P, G174K, S175P, S27K, S173P, G174K, S175P, F180Y, 0.96 1.08 F180Y, G182A, L184F, Q198E, V286R, G182A, L184F, Q198E, V286R, T297P T297P N87K S27K, N87K, S171D, S173P, G174R, S27K, S171D, S173P, G174R, S175P, 1.72 1.01 S175P, F180Y, G182A, L184F, F180Y, G182A, L184F, Q198E, Q198E, T297P T297P N87K S27K, N87K, V162T, S171D, S173P, S27K, V162T, S171D, S173P, G174R, 1.17 0.98 G174R, S175P, F180Y, G182A, L184F, S175P, F180Y, G182A, L184F, Q198E, S274V, T297P Q198E, S274V, T297P N87K S27K, N87K, S171D, S173P, G174R, S27K, S171D, S173P, G174R, 0.96 1.07 S175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P N87K S27K, N87K, S171D, S173P, G174R, S27K, S171D, S173P, G174R, 1.03 1.09 S175P, F180Y, Q198E, T297P S175P, F180Y, Q198E, T297P N87K S4K, S27K, N87K, E127Q, G132H, S4K, S27K, E127Q, G132H, Y156R, 1.16 NA Y156R, S173P, S175P, F180Y, Q198E, S173P, S175P, F180Y, Q198E, T297P T297P S129K S27K, S114V, I121V, S129K, S173P, S27K, S114V, I121V, S173P, G174K, 1.05 1.15 G174K, S175P, F180Y, L184F, S175P, F180Y, L184F, Q198E, N199K, Q198E, N199K, T297P T297P T24R T24R, S173P, S175P, F180Y S173P, S175P, F180Y 1.87 1.26

(252) TABLE-US-00005 TABLE 5 AMSA relative wash performance.sub.mutation for the mutations in column 1, using a standard full egg with pigment textile swatch (CS-37). Variants with Variants without Mutation the mutations mutations CS-37 Q18H Q18H, S173P, S175P S173P, S175P 1.39 G112R G112R, S173P, S175P, S173P, S175P, F180Y 3.56 F180Y G112R G112R, S173P, S175P S173P, S175P 2.49 D116A D116A, S173P, S175P, S173P, S175P, F180Y 1.81 F180Y T131H T131H, S173P, S175P S173P, S175P 1.06 G142K G142K, S173P, S175P S173P, S175P 3.68 G142R G142R, S173P, S175P S173P, S175P 5.80 S143H S143H, S173P, S175P S173P, S175P 1.10 D147W D147W, S173P, S175P S173P, S175P 1.68 G174H I137E, S173P, G174H, I137E, S173P, S175P, 2.41 S175P, F180Y, T297P F180Y, T297P D206H S173P, S175P, D206H S173P, S175P 1.07 F207H S173P, S175P, F207H S173P, S175P 1.69 I220H S173P, S175P, I220H S173P, S175P 1.42 I247K S173P, S175P, I247K S173P, S175P 1.31 N268H S173P, S175P, N268H S173P, S175P 1.33 T269H S173P, S175P, T269H S173P, S175P 1.05 T278H S173P, S175P, T278H S173P, S175P 1.17