Patent

1. An isolated subtilisin variant having protease activity, wherein said variant comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 18, wherein said variant comprises the amino acid substitutions that correspond to substitutions S039E-S099R-F128A in the polypeptide of SEQ ID NO:85, and wherein said variant has one or more improved properties compared to the reference subtilisin having the amino acid sequence of SEQ ID NO:85 selected from the group consisting of improved protease activity, improved cleaning activity and improved thermostability in the presence of a detergent.

2. The isolated subtilisin variant of claim 1 , wherein said variant comprises one or more amino acid substitutions that correspond to substitutions in the polypeptide of SEQ ID NO:85 selected from:

A037T-S039E-N042T-S099R-V102I-T114Q-S126T-F128A-N242D;

A037T-S039E-S099R-V102I-T114Q-S126T-F128A-R143A-N212S-N242D;

A037T-S039E-N074D-S099R-V102I-S126T-F128A-R143A-N212S-N242D;

A037T-S039E-N042T-N074D-S099R-V102I-S126T-F128A-S158T-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-5099R-T114A-F128A;

A037T-S039E-I043V-A047V-T055G-5099R-V102I-T114Q-S126T-F128A-N212S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-5099R-T114Q-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-5099R-T114A-F128A-N242D;

A037T-S039E-A047V-I080V-N0855-E087D-5099R-T114Q-F128A-R143A-N242D;

S036A-S039E-I043V-A047V-T055M-I080V-N085S-E087D-5099R-T114A-F128A;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-5099R-V102I-T114Q-F128A;

A037T-S039E-I043V-A047V-N074D-N0855-E087D-5099R-T114A-F128A-S158T-N212S;

A037T-S039E-N074D-I080V-N085S-E087D-5099R-V102I-T114Q-S126T-F128A-R143A;

A037T-S039E-I043V-A047V-T055G-N074D-I080V-5099R-V102I-T114Q-S126T-F128A;

A037T-S039E-I043V-A047V-N074D-I080V-5099R-V102I-T114Q-S126T-F128A-N242D;

R027K-A037T-S039E-A047V-T055G-T056Y-N074D-5099R-V102I-S126T-F128A-N242D;

A037T-S039E-N042T-A047V-N074D-I080V-N0855-E087D-5099R-T114Q-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N0855-E087D-5099R-T114A-S126T-F128A-N212S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-5099R-T114A-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N0855-E087D-5099R-T114Q-S126T-F128A-R143A;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-5099R-V102I-T114A-F128A-N212S;

A037T-S039E-N042T-A047V-I080V-N0855-E087D-5099R-V102I-T114Q-F128A-N242D;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-5099R-T114A-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114Q-F128A-N212S-N242D;

A037T-S039E-A047V-I080V-N085S-E087D-S099R-V102I-T114Q-F128A-R143A-N242D;

A037T-S039E-I043V-A047V-T055G-I080V-N085S-E087D-S099R-T114Q-S126T-F128A;

R027K-A037T-S039E-A047V-I080V-N085S-E087D-S099R-T114Q-F128A-N212S-N242D;

T009S-A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-V1971;

S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-N212S-K245L-N246S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-I190L-A224V;

S036A-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-N121S-F128A-R143Q;

S036A-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-N212S-S255N;

S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-R143Q-N242D-N246K;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-S126T-F128A-N212S;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-F128A-N212S;

A037T-S039E-N042T-A047V-I080V-N085S-E087D-S099R-T114A-F128A-S158T-N212S;

V004I-A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-N182S;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-S126T-F128A-S 158T-N212S;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-F128A-S 158T-N212S;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-F128A-N212S-N242Q-K245L;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-F128A-N212S-N242Q;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-F128A-N212S-K245L;

A037T-S039E-A047V-S099R-V102I-T114A-1119V-S126T-F128A-R143A-S158T-G160S-N212S;

A037T-S039E-N042T-A047V-T056Y-N074D-I080V-N085S-E087D-S099R-T114Q-S126T-F128A;

S024F-A037T-S039E-A047V-N074D-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-N212S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-R143A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A-A237T-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-T055G-I080V-N085S-E087D-S099R-V102I-T114Q-F128A-R143A;

A037T-S039E-N042T-A047V-T055G-I080V-N085S-E087D-S099R-T114Q-F128A-N212S-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114Q-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-S099R-T114A-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-R143A;

A037T-S039E-I043V-A047V-T055G-I080V-N085S-E087D-S099R-T114Q-S126T-F128A;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-F128A-S 158T-N212S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A-A237T-N242D;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-S126T-F128A-N242D;

R027K-A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-S099R-T114A-S126T-F128A-R143A;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-N121S-F128A-N246S-S-255N;

A037T-S039E-A047V-I080V-N085S-E087D-S099R-T114Q-F128A-R143A-N242D;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-F128A-S158T-N212S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114Q-I119V-S126T-F128A;

A037T-S039E-I043V-A047V-T055G-I080V-N085S-E087D-S099R-V102I-T114A-S126T-F128A-S158T;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A-R143A-S158T-N242D;

A037T-S039E-N042T-I043V-R044S-A047V-N074D-I080V-N085S-E087D-S099R-T114A-S126T-F128A;

A037T-S039E-N042T-I043V-R044S-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-V102I-T114A-I119V-S126T-F128A-F257Y;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-V102I-T114A-N121S-F128A-R143Q;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-S126T-F128A-S 158T-N212S;

S036A-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-F128A-A224V-S255N;

A037T-S039E-I043V-A047V-T055G-N074D-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-T055G-I080V-N085S-E087D-5099R-V102I-T114Q-S126T-F128A-R143A;

A037T-S039E-I043V-A047V-I080V-N0855-E087D-5099R-V102I-T114Q-S126T-F128A-R143A-N242D;

A037T-S039E-I043V-A047V-T056Y-I080V-N0855-E087D-5099R-T114Q-S126T-F128A-S158T-N242D;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-5099R-T114Q-I119V-S126T-F128A-F257Y;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-5099R-T114Q-I119V-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-N074D-I080V-N0855-E087D-5099R-T114A-S126T-F128A-S158T-N212S-N242Q;

A037T-S039E-I043V-A047V-N074D-I080V-N0855-E087D-5099R-T114A-S126T-F128A-T188A-I190L-F257Y;

V004I-T0095-A037T-S039E-I043V-A047V-I080V-N0855-E087D-5099R-T114A-F128A-A224V-K245L-S255N;

A037T-S039E-N042T-A047V-T056Y-N074D-I080V-N0855-E087D-5099R-T114Q-S126T-F128A;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-S126T-F128A-S 158T-N212S-N242Q;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-S126T-F128A-S 158T-N212S-K245L;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114P-S126T-F128A-R143T-S158T-N212K-N242D;

A037T-S039E-I043V-A047V-T056Y-N074D-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-R143A-N242D;

A037T-S039E-I043V-A047V-T055G-T056Y-N074D-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A;

S036A-A037N-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-N242D-K245L-N246S-S255N;

A037T-S039E-I043V-A047V-N074D-I080V-N085S-E087D-S099R-T114A-S126T-F128A-S158T-N212S-N242Q-K245L;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-1119V-S126T-F128A-S158T-G160S-N242D-F257Y;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-N121S-F128A-N182S-I190L-K245L-N246S-S255N;

A037T-S039E-N074D-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-R143A;

A037T-S039E-I043V-A047V-T055G-N074D-I080V-S099R-V102I-T114Q-S126T-F128A;

A037T-S039E-I043V-A047V-N074D-I080V-S099R-V102I-T114Q-S126T-F128A-N242D;

A037T-S039E-A047V-N074D-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-S158T-N242D;

A037T-S039E-N042T-A047V-N074D-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-N242D;

A037T-S039E-I043V-A047V-N074D-N085S-E087D-S099R-T114A-S126T-F128A-S 158T-N212S-N242Q-K245L;

T009S-S036A-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-R143Q-N182S-T188A-I190L;

A037T-S039E-I043V-A047V-T055G-N074D-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-S158T-N242D;

A037T-S039E-I043V-A047V-T055G-N074D-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-R143A-S158T-N212S;

A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-F128A-R143Q-N212S-A224V-Y232N-K245L-N246S-S255N;

V004I-T009S-A037T-S039E-I043V-A047V-I080V-N085S-E087D-S099R-T114A-N121S-F128A-R143Q-A2241-R231K-K245L;

R027K-A037T-S039E-A047V-I080V-N085S-E087D-S099R-T114Q-I119V-S126T-F128A-G160S-N242D-F257Y;

R027K-A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-S 158T-G160S-N242D-F257Y;

R027K-A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-S099R-V102I-T114Q-S126T-F128A-T188A-I190L-F257Y;

R027K-A037T-S039E-I043V-A047V-T056N-I080V-N0855-E087D-5099R-T114P-S126T-F128A-R143T-V1591-N212T-F257Y;

A037T-S039E-I043V-A047V-T056Y-I080V-N085S-E087D-5099R-T114Q-I119V-S126T-F128A-S 158T-V1591-G1605-F257Y;

A037T-S039E-I043V-A047V-T055G-T056Y-N074D-I080V-N0855-E087D-5099R-V102I-T114Q-S126T-F128A-N242D;

R027K-A037T-S039E-A047V-T056Y-N074D-I080V-N0855-E087D-5099R-T114Q-I119V-S126T-F128A-N242D-F257Y;

A037T-S039E-N042T-N074D-S099R-V102I-S126T-F128A-S158T-N242D;

R027K-A037T-S039E-A047V-T055G-T056Y-N074D-5099R-V102I-S126T-F128A-N242D;

S039E-I043V-A047V-I080V-N085S-E087D-5099R-T114A-F128A-A224V-N242D-N2465-S255N;

A037T-S039E-I043V-A047V-N074D-I080V-N0855-E087D-5099R-V102I-T114A-F128A-R143Q-S158T-A224V-S255N;

A037T-S039E-I043V-A047V-P0545-T056Y-A057Q-N074D-I080V-N0855-E087D-5099R-T114A-N121S-F128A-S158T-A224V-S255N;

A037T-S039E-N042T-I043V-R0445-A047V-N074D-I080V-N0855-E087D-5099R-V102I-S126T-F128A-S158T-G160S-N242D;

S039E-T056Y-S099R-F128A;

S039E-S099R-T114Q-F128A;

S039E-N085S-S099R-F128A;

S039E-E087D-S099R-F128A;

S039E-S099R-S126T-F128A;

S039E-E087D-S099R-S126T-F128A;

S039E-T056Y-S099R-S126T-F128A;

S039E-E087D-5099R-F128A-N242D;

A037T-S039E-E087D-5099R-T114A-F128A;

S039E-T056Y-E087D-S099R-S126T-F128A;

S039E-5099R-T114Q-S126T-F128A-N242D;

A037T-S039E-5099R-T114Q-S126T-F128A-N242D;

A037T-S039E-T056Y-S099R-S126T-F128A-N242D;

A037T-S039E-T056Y-N085S-E087D-S099R-T114Q-F128A;

A037T-S039E-A047V-T056Y-5099R-T114Q-S126T-F128A;

A037T-S039E-A047V-T056Y-5099R-T114A-S126T-F128A;

A037T-S039E-T056Y-S099R-T114Q-S126T-F128A-N242D;

S039E-A047V-E087D-S099R-T114Q-S126T-F128A-N242D;

S039E-A047V-T056Y-N085S-E087D-S099R-T114Q-S126T-F128A;

S039E-A047V-I080V-E087D-S099R-T114A-S126T-F128A-N242D;

A037T-S039E-T056Y-N085S-S099R-T114Q-S126T-F128A-N242D;

S039E-A047V-T056Y-E087D-S099R-T114A-S126T-F128A-N242D;

S039E-A047V-T056Y-E087D-S099R-T114Q-S126T-F128A-N242D;

A037T-S039E-A047V-I080V-N085S-E087D-S099R-T114A-S126T-F128A;

A037T-S039E-I080V-N085S-E087D-S099R-T114Q-S126T-F128A-N242D;

A037T-S039E-T056Y-N085S-E087D-S099R-T114A-S126T-F128A-N242D;

A037T-S039E-A047V-T056Y-E087D-S099R-T114A-S126T-F128A-N242D; and

combinations thereof.

3. The isolated subtilisin variant of claim 1 , wherein the improved property is

(i) improved protease activity, wherein said variant has a performance index (PI) on N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (N-suc-AAPF-pNA) or dimethyl casein substrate;

(ii) improved cleaning performance in the presence of a detergent, wherein said variant has a blood/milk/ink (BMI) and/or egg stain cleaning PI >1; and/or

(iii) improved thermostability in the presence of a detergent, wherein said variant has a stability PI >1.

4. A composition comprising the subtilisin variant of claim 1 .

5. The composition of claim 4 , wherein said composition is a detergent composition.

6. The composition of claim 5 , wherein the detergent composition is selected from a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent.

7. The composition of claim 4 , wherein said composition further comprises calcium ions and/or zinc ions; one or more enzyme stabilizers; about 0.001% to about 1.0 weight % of said variant; one or more bleaching agents; one or more adjunct materials; and/or one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, additional serine proteases, and combinations thereof.

8. The composition of claim 4 , wherein said composition is phosphate-free.

9. The composition of claim 4 , wherein said composition is a granule, powder, solid, bar, liquid, tablet, gel, paste or unit dose composition.

10. The composition of claim 4 , wherein said composition is boron free.