US2018216092A1PendingUtilityA1
Compositions and methods comprising serine protease variants
Est. expiryMay 6, 2030(~3.8 yrs left)· nominal 20-yr term from priority
Inventors:Viktor Yuryevich AlekseyevNeelam S. AminLuis G. Cascao-PereiraKatherine D. CollierDavid A. EstellJames T. Kellis, Jr.Ayrookaran J. PoulosePhilip Frank SouterGlenn Steven WardKatherine AugustynJian Yao
C12Y 304/21062C12N 15/63C12N 9/54C11D 3/386C11D 3/38681
66
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Claims
Abstract
The present invention provides serine protease variants produced there from. Specifically, the present invention provides serine protease variants having one or more substitutions as compared to a reference serine protease. In addition, the present invention provides compositions comprising these serine protease variants. In some embodiments, the present invention provides cleaning compositions comprising at least one of these serine protease variants.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-N043R, N062E-A158E, S103G-A158E, S128N-A158E, A016S-A158E, V104L-A158E, E089P-A158E, L111V-A158E, T022A-A158E, S101A-A158E, L148I-A158E, P129E-A158E, T022A-E089P, A016S-E089P, N062E-E089P, N062E-E271F, A158E-E271F, R186H-E271F, P129E-E271F, L111V-E271F, Y209E-E271F, A016S-E271F, S188D-E271F, T022A-E271F, G159E-E271F, V104L-E271F, S101A-E271F, E089P-E271F, S128N-E271F, S103G-E271F, L148I-E271F, H249R-E271F, N062E-G159E, A016S-G159E, S128N-G159E, L148I-G159E, L111V-G159E, E089P-G159E, T022A-G159E, P129E-G159E, S103G-G159E, V104L-G159E, A158E-G159E, S101A-G159E, A158E-H249R, L111V-H249R, P129E-H249R, N062E-H249R, A016S-H249R, R186H-H249R, L148I-H249R, G159E-H249R, S101A-H249R, S188D-H249R, V104L-H249R, Y209E-H249R, T022A-H249R, S128N-H249R, S103G-H249R, E089P-H249R, T022A-L111V, S101A-L111V, A016S-L111V, V104L-L111V, N062E-L111V, S103G-L111V, E089P-L111V, A016S-L148I, N062E-L148I, T022A-L148I, P129E-L148I, V104L-L148I, S103G-L148I, S128N-L148I, S101A-L148I, E089P-L148I, L111V-L148I, A016S-N062E, T022A-N062E, N062E-P129E, T022A-P129E, S128N-P129E, A016S-P129E, S101A-P129E, V104L-P129E, E089P-P129E, S103G-P129E, L111V-P129E, N062E-R186H, S128N-R186H, S101A-R186H, T022A-R186H, A016S-R186H, A158E-R186H, E089P-R186H, P129E-R186H, G159E-R186H, S103G-R186H, V104L-R186H, L111V-R186H, L148I-R186H, N062E-S101A, T022A-S101A, A016S-S101A, E089P-S101A, N062E-S103G, T022A-S103G, A016S-S103G, S101A-S103G, E089P-S103G, N062E-S128N, A016S-S128N, T022A-S128N, S101A-S128N, V104L-S128N, E089P-S128N, S103G-S128N, L111V-S128N, L111V-S188D, N062E-S188D, A016S-S188D, L148I-S188D, T022A-S188D, S128N-S188D, S101A-S188D, V104L-S188D, E089P-S188D, P129E-S188D, G159E-S188D, R186H-S188D, S103G-S188D, A158E-S188D, A016S-T022A, A016S-V104L, T022A-V104L, S101A-V104L, N062E-V104L, S103G-V104L, E089P-V104L, G159E-Y209E, L111V-Y209E, S101A-Y209E, A016S-Y209E, S128N-Y209E, L148I-Y209E, P129E-Y209E, N062E-Y209E, T022A-Y209E, S103G-Y209E, A158E-Y209E, S188D-Y209E, V104L-Y209E, E089P-Y209E, R186H-Y209E, N018R-W241R, G020R-W241R, S024R-W241R, S009A-W241R, G020R-W241R, V004R-W241R, N043R-W241R, S078R-W241R, T022R-W241R, G115R-W241R, A001R-W241R, S212F-W241R, L082R-W241R, N018R-V244R, S024R-V244R, S078R-V244R, G020R-V244R, S212F-V244R, S009A-V244R, L082R-V244R, A001R-V244R, N043R-V244R, T022R-V244R, V004R-V244R, G115R-V244R, W241R-V244R, S242R-V244R, A001R-V004R, S009A-T022R, N018R-T022R, G020R-T022R, V004R-T022R, A001R-T022R, S024R-S242R, N018R-S242R, V004R-S242R, G020R-S242R, S212F-S242R, L082R-S242R, S078R-S242R, A001R-S242R, S009A-S242R, T022R-S242R, G115R-S242R, N043R-S242R, W241R-S242R, N018R-S212F, T022R-S212F, V004R-S212F, S024R-S212F, A001R-S212F, G115R-S212F, G020R-S212F, S009A-S212F, N043R-S212F, S078R-S212F, L082R-S212F, S009A-S078R, G020R-S078R, S024R-S078R, T022R-S078R, N018R-S078R, V004R-S078R, A001R-S078R, N043R-S078R, T022R-S024R, G020R-S024R, N018R-S024R, A001R-S024R, V004R-S024R, S009A-S024R, V004R-S009A, A001R-S009A, S242R-N269R, S024R-N269R, G020R-N269R, T022R-N269R, H249R-N269R, S212F-N269R, N043R-N269R, V244R-N269R, A001R-N269R, N018R-N269R, S078R-N269R, S009A-N269R, G115R-N269R, W241R-N269R, V004R-N269R, L082R-N269R, N018R-N043R, G020R-N043R, V004R-N043R, T022R-N043R, S009A-N043R, A001R-N043R, S024R-N043R, S009A-N018R, V004R-N018R, A001R-N018R, S024R-L082R, S009A-L082R, N018R-L082R, A001R-L082R, S078R-L082R, G020R-L082R, T022R-L082R, V004R-L082R, N043R-L082R, N043R-H249R, G020R-H249R, V004R-H249R, N018R-H249R, S009A-H249R, S212F-H249R, T022R-H249R, S024R-H249R, G115R-H249R, A001R-H249R, L082R-H249R, S242R-H249R, W241R-H249R, V244R-H249R, S078R-H249R, N018R-G115R, G020R-G115R, T022R-G115R, S078R-G115R, S009A-G115R, V004R-G115R, A001R-G115R, L082R-G115R, N043R-G115R, S024R-G115R, S009A-G020R, N018R-G020R, V004R-G020R, A001R-G020R, S009A-E271L, G020R-E271L, S024R-E271L, V244R-E271L, W241R-E271L, N043R-E271L, T022R-E271L, H249R-E271L, S212F-E271L, G115R-E271L, S242R-E271L, S078R-E271L, V004R-E271L, N269R-E271L, A001R-E271L, N018R-E271L, and L082R-E271L, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
2 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-N043R, G020K-N062E, S024F-N116L, G020K-S024F, S024R-A174T, S024R-G118R, S024R-K235F, S024R-P086R, S024R-P086W, S078R-G118R, T033S-G118R, T033S-K235F, Y209A-W241R, G020R-N076D, N018R-Q245R, S024R-R045T, A232V-Q245R, G118R-A172V, G118R-A194T, I008T-S024R, K235F-N243F, N018R-S103A, N018R-V104I, P086W-G118R, P086W-N243F, P086W-Y209A, S024C-T033S, S024R-A232V, S024R-N243F, S024R-P239Q, S024R-S101G, S024R-S141G, S024R-T033S, S024R-T274I, S024R-Y209A, S078R-P086W, S101G-A232V, T033S-L148F, T033S-P086W, T033S-P201S, T033S-S078R, T033S-W241R, T033S-Y209A, A230E-H249R, A232V-H249R, G118R-K235F, N076D-Q245R, P086W-K235F, S024R-R247H, S024R-V104A, S078R-K235F, S101G-H249R, S103A-A232V, T033S-A048T, T033S-P239T, T033S-T253A, T143A-Y209A, Y209A-K235F, N018R-R045T, Y209A-N243F, S024R-A272P, S024R-R269C, S101G-V104I, V104I-A232V, N076D-H249R, and S024R-N076D, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
3 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-N076D, S024R-R045T, A230E-H249R, N018R-R045T, N018R-Q245R, S101G-A232V, S024R-A232V, A232V-Q245R, S024R-S101G, N018R-V104I, N018R-S103A, S101G-H249R, A232V-H249R, S103A-A232V, N076D-Q245R, S101G-V104I, V104I-A232V, N076D-H249R, S024R-N076D, S024F-N116L, G020K-S024F, G020K-N062E, T033S-G118R, S024R-P086W, S024R-G118R, S024R-P086R, Y209A-W241R, S024R-W241R, S024R-K235F, G118R-Y209A, S078R-G118R, T033S-K235F, S024R-A174T, P086W-Y209A, I008T-S024R, P086W-G118R, T033S-W241R, S024R-N243F, S024R-Y209A, T033S-P086W, S024R-T033S, P086W-N243F, T033S-P201S, S024R-P239Q, S078R-P086W, K235F-N243F, G118R-A172V, T033S-L148F, T033S-S078R, T033S-N243F, S024C-T033S, G118R-A194T, T033S-Y209A, S024R-S141G, S024R-T274I, P086W-K235F, A015T-T033S, Y209A-K235F, S024R-R247H, S078R-K235F, S024R-V104A, T033S-A048T, G118R-K235F, T033S-T253A, T143A-Y209A, T033S-P239T, Y209A-N243F, S024R-A272P, and S024R-R269C, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
4 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: G020K-G023A-N043W-G118R-S128I-P129E-G159D-S188D, G020K-G023A-N062E-N116L, G020K-G023A-N062E-N116L-S188D, G020K-G023A-N062E-N116L-S188D-T213A, G020K-G100S-N116L-A158E-S166D-N243F, G020K-N043W-N062E-N116L-S188D, G020K-N062E, G020K-N062E-N116L, G020K-N062E-N116L-S188D, G020K-N062E-N116L-T213A, G020K-N062E-S188D-T213A, G020K-S024F-N062E-N116L-G118R-S188D, G020K-S024F-N062E-N116L-S188D, G020K-S024F-N062E-S188D-T213A, G023A-N062E-N116L-S188D, G023A-S024F-N062E-N116L-T213A, N043R-N076D-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R-E271F, N043R-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, N043W-N062E-N116L, N043W-S101D-S212M-N243F, N062E-N116L-G118R-S188D, N062E-N116L-G118R-T213A, N062E-N116L-S188D-T213A, S024F-G118R-S128I-P129E-G159D, S024F-N062E-N116L-S188D, S024R-S101G-S103A-V104I-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-S130A-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S130A-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S101D-S103N-N116L-S144R-A215D, S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-N043R-S101G-S103A-V104I-G159D-S188D-L217E-A232V-Q245R-N248D-E271F, T022A-N043R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-A232V-N248D-E271F, T022A-N043R-S103A-V104I-S128I-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-A232V-N248D, T022A-S101G-S103A-V104I-G159D-L217E-A232V-Q245R-N248D-E271F, T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022L-T038F-A048R-N062E-G100S-R186K, T033S-N043W-N218D-P239G-N243F, V026F-A048R-S105T-T213A-N218D-T224A, V026F-L031F-S078N-G102A-S160D, V026F-V051W-V104L-S106E, V104L-S105T-T213A-L217E-S256N, A016S-N062E-A158E-H249R, A016S-N062E-A158E-R186H-E271F, A016S-N062E-A158E-R186H-H249R, A016S-N062E-V104L-A158E-R186H-E271F, A016S-S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, G020K-G023A-N062E-S188D, G020K-G023A-S024F-N062E-G118R-S188D-T213A, G020K-N043W-N062E-N116L-S188D-T213A-P239G, G020K-S024F-N062E-S188D-P239G, G023A-N062E-N116L-G118R, G023A-N062E-N116L-G118R-S188D-P239G, G023A-S024F-N062E-N116L-G118R, H017R-T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, N043R-N076D-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, N062E-A158E-G159E-H249R, N062E-A158E-H249R, N062E-A158E-R186H-E271F, N062E-A158E-R186H-H249R, N062E-A158E-R186H-H249R-E271F, N062E-A158E-S188D-H249R, N062E-A158E-S188D-H249R-E271F, N062E-G159E-H249R, N062E-G159E-H249R-E271F, N062E-G159E-R186H-H249R, N062E-G159E-S188D-H249R, N062E-R186H-S188D-H249R-E271F, N062E-S101A-A158E-H249R, N062E-S101A-A158E-R186H-E271F, N062E-S101A-A158E-R186H-H249R-E271F, N062E-S101A-G159E-H249R, N062E-S101A-R186H-H249R, N062E-S101A-S188D-H249R, N062E-S101A-S188D-H249R-E271F, N062E-S101A-V104L-A158E-R186H-E271F, N062E-S128N-A158E-G159E-E271F, N062E-V104L-A158E-S188D-H249R-E271F, N076D-S101G-S103A-V104I-A232V-M222Q-Q245R, S024F-N062E-N116L-P239G, S024F-N062E-S188D-T213A, S024F-N116L, S024R-N043R-N076D-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-A158E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-G159E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-L148I-A158E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-L148I-A158E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-L148I-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-G159E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-L148I-A158E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-A158E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-S128N-P129E-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-P129E-A232V-Q245R-N248D, S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F-E271F, S101A-S103A-V104L-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S128N-A158E-R186H-E271F, S101A-S128N-A158E-Y209E-H249R, S101A-V104L-A158E-R186H-S188D-H249R, S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-S188D-M222Q-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-S188D-M222S-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A232V-M222Q-Q245R, S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S188D-A232V-Q245R-N248D-H249R, S101G-S103G-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103S-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103G-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103G-V104V-A158E-S188D-A232V-Q245R-N248D-H249R, S128N-A158E-G159E-E271F, S128N-A158E-H249R, S128N-A158E-R186H-E271F, S128N-A158E-R186H-H249R, S128N-A158E-R186H-S188D-E271F, S128N-A158E-S188D-E271F, S128N-A158E-S188D-H249R, S128N-A158E-S188D-Y209E-E271F, S128N-A158E-Y209E-, T022A-N043R-S103A-V104I-G118R-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-N062E-A158E, T022A-S024R-S101D-S103A-V104I-G118R-G159D-S188D-A232V-N248D-E271F, T022A-S024R-S101D-S103A-V104I-G118R-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-S101D-S103A-V104I-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-A158E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-P129E-A158E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-P129E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-S188D-A232V-Q245R-N248D, T022A-S024R-S103A-V104I-G118R-G159D-S188D-L217D-A232V-N248D, T022A-S024R-S103A-V104I-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-A158E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-A158E-G159E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-P129E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-P129E-S188D-A232V-N238R-Q245R-N248D, T022A-S128N-A158E-H249R, V104L-S128N-A158E-H249R, V104L-S128N-A158E-R186H-E271F, V104L-S128N-A158E-R186H-H249R, and V104L-S128N-A158E-S188D-H249R, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
5 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: A016S-N062E-A158E-H249R-E271F, A016S-N062E-S128N-R186H-E271F, A016S-N062E-V104L-R186H-S188D-E271F, A016S-S101A-S128N-R186H, A016S-S128N-A158E-R186H, A016S-V104L-A158E-R186H-E271F, A016S-V104L-S188D-H249R, A158E-R186H-H249R, A158E-R186H-S188D-H249R-E271F, G020K-G023A-N116L-S188D, G020K-G023A-S024F-N062E-N116L-S188D-T213A, G020K-N043W-N062E-N116L-P239G, G020K-S024F-N043W-N062E-N116L-T213A, G020K-S024F-N062E, G023A-N043W-N062E-N116L-G118R-T213A, G023A-T038F-S078N-G100S-S212M-A215D, G100S-N116L-A158E-T213A, G102A-S103N-S105T-A194E, H017R-T022A-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, N043R-N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, N043R-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R, N043W-N062E-N116L-S188D, N062E-A158E-H249R-E271F, N062E-A158E-R186H-S188D-H249R, N062E-L148I-G159E, N062E-N116L-S188D, N062E-R186H-H249R, N062E-S078N-G102A-N116L-S144R-L250I, N062E-S101A-A158E-R186H-H249R, N062E-S101A-A158E-S188D-H249R, N062E-S101A-R186H, N062E-S101A-R186H-E271F, N062E-S101A-R186H-H249R-E271F, N062E-S101A-R186H-S188D-E271F, N062E-S101A-R186H-S188D-H249R, N062E-S101A-V104L-R186H-S188D-E271F, N062E-S128N-A158E, N062E-S128N-G159E-H249R, N062E-S188D-P239G, N062E-V104L-G159E-H249R, N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, N076D-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, N076D-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, N076D-S101G-S103A-V104I-A114V-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, N076D-S101G-S103A-V104I-A232V-M222S-Q245R, S024F-A048R-G118R-S166D-L217E, S024F-N062E-N116L, S024F-N062E-N116L-T213A-P239G, S024F-N062E-S188D-P239G, S024F-S101D-G118R-A215D-L250I-A272F, S024R-K027R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S024R-S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-G159E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-L148I-A158E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-L148I-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-G159E-S188D-A232V-Q245R-N248D, S078N-V104L-G118R-S128D, S101A-A158E-R186H-H249R, S101A-A158E-R186H-S188D-H249R, S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R-E271F, S101A-S103G-A158E-R186H-H249R, S101A-S103S-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103S-V104I-G159E-A232V-Q245R-N248D-H249R, S101A-S128N-A158E-H249R, S101A-S128N-A158E-S188D-Y209E-E271F, S101A-S128N-A158E-Y209E, S101A-S128N-P129E-R186H-H249R, S101A-V104L-A158E-R186H-H249R, S101A-V104L-A158E-R186H-S188D-E271F, S101A-V104L-S128N-A158E-R186H-E271F, S101G-S103A-V104I-A158E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-A232V-M222S-Q245R, S101G-S103A-V104I-P129E-A158E-G159E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-A158E-S188D-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S128L-S130A-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S128N-P129E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-A232V-Q245R-N248D, S101G-S103A-V104I-S130A-A158E-N183D-S188D-L217E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S188D-A232V-N238R-Q245R-N248D, S101G-S103A-V104L-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103A-V104I-G159E-A232V-Q245R-N248D-H249R, S101S-S103S-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103S-V104V-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-N043R-N062E-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-N043R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-N043R-S101D-S103A-V104I-G118R-P129E-G159D-S188D-A232V-N248D-E271F, T022A-N043R-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, T022A-N043R-S103A-V104I-G118R-G159D-S188D-A232V-N248D-E271F, T022A-N043R-S103A-V104I-G118R-G159D-S188D-L217D-A232V-N248D-E271F, T022A-N043R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-A232V-N248D, T022A-N043R-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-S024R-N043R-S101D-S103A-V104I-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-N043R-S103A-V104I-G159D-S188D-L217D-A232V-N248D-E271F, T022A-S024R-S101D-S103A-V104I-G118R-S128I-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-L148I-A158E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-L148I-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S103A-V104I-G118R-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S024R-S103A-V104I-G159D-S188D-L217D-A232V-N248D-E271F, T022A-S024R-S103A-V104I-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, T022A-S101A-A158E-R186H-H249R, T022A-S101G-S103A-V104I-A158E-G159E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-H249R-E271F, T022A-S101G-S103A-V104I-G159E-S188D-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-G159E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S103A-V104I-G118R-G159D-S188D-L217D-A232V-Q245R-N248D, T022A-S103A-V104I-G159D-S188D-A232V-N248D, T022L-L031F-G102A-S128D-T224A-N243F, T022L-T038F-V121F-S160D-A272F, V026F-S078N-G159C-R186K-N243F, V104L-A158E-H249R, V104L-A158E-R186H-H249R, V104L-A158E-R186H-H249R-E271F, V104L-A158E-R186H-S188D-H249R, V104L-A158E-R186H-S188D-H249R-E271F, V104L-A158E-S188D-H249R-E271F, V104L-S128N-A158E-R186H-S188D-E271F, V104L-S128N-G159E-E271F, V104L-S128N-R186H-S188D-H249R, and V121F-N185E-T224A-P239G, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
6 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: G118R-Y209A, G118R-Y209A-N243F, L021M-S024R-T033S, P005S-S078R-G118R-W241R, P086W-G118R-A133V, P086W-G118R-N243F, P086W-G118R-Y209A, P086W-Y209A-N243F, S024R-A174T, S024R-G118R, S024R-G118R-Y209A, S024R-G118R-Y209A-K235F, S024R-G118R-Y209A-N243F, S024R-K235F, S024R-P086R, S024R-P086S-S141G, S024R-P086W, S024R-P086W-G118R, S024R-P086W-G118R-V203I, S024R-P086W-N243F, S024R-P086W-Y209A, S024R-S078R-P086W, S024R-S078R-P086W-G118R-A270T, S024R-S078R-P086W-N243F, S024R-T033S-A133V, S024R-T033S-G118R, S024R-T033S-P086S-S085N-K235F, S024R-T033S-P086S-S087N-Y209A, S024R-T033S-P086W, S024R-T033S-P086W-G118R, S024R-T033S-S078R-P086W, S024R-T033S-S078R-P086W-G118R, S024R-T033S-S078R-Y209A, S024R-T033S-W241R, S024R-T033S-Y209A-K235F, S024R-Y209A-W241R, S078R-G118R, S078R-P086W-K235F, S078R-P086W-N243F, S078R-P086W-Y209A, S078R-Y209A-K235F, T033S-A172V-Y209A, T033S-G118R, T033S-G118R-G159D-Y209A, T033S-G118R-N243F, T033S-G118R-W241R, T033S-G118R-Y209A-N243F, T033S-K235F, T033S-P086W-G118R, T033S-P086W-G118R-Y209A-N243F, T033S-P086W-N243F, T033S-S078R-P086W, T033S-S078R-P086W-G118R-Y209A, T033S-S078R-Y209A, T033S-Y209A-N243F, Y209A-W241R, A001R-S009A-N043R, A001R-S101G-S103A-V104I-A232V-Q245R, A001T-N018R-S024R-N076D-N116A-T213A-H249R, A194F-G211Q-Q236N, E089I-N116A-N117F-T224A-H249R, G020R-G025R-N116A-Y167W, G020R-H249R-N269R, G020R-N043D-N076D-A230E-H249R, G020R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-N043D-S101G-S103A-V104I-A232V-Q245R, G020R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-N043R-A230E, G020R-N043R-A230E-S242R, G020R-N043R-E271L, G020R-N043R-H249R, G020R-N043R-H249R-E271L, G020R-N043R-N269R, G020R-N043R-R045T-A230E, G020R-N043R-R045T-S101A-N269R, G020R-N043R-R045T-S242R, G020R-N043R-S101A-N116A-T213A-A215F, G020R-N043R-S101A-N269R, G020R-N043R-S101G-S103A-V104I-A232V-Q245R, G020R-N043R-S212F, G020R-N043R-S212F-W241R, G020R-N043R-S242R, G020R-N043R-S242R-E271L, G020R-N043R-S242R-H249R, G020R-N043R-V244R, G020R-N043R-W241R, G020R-N062Q-E089I-R186K-S212M, G020R-N076D, G020R-N076D-S101G-S103A-V104I-A232V-Q245R, G020R-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-N116A-N269R, G020R-P052N-N062Q-Y091F-Y192W, G020R-Q059A-S144R-Y192W-T224A, G020R-S024R-K27E-N043R-N076D-A230E, G020R-S024R-N043D-H249R, G020R-S024R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-S024R-N043D-S242R, G020R-S024R-N043R-R045T, G020R-S024R-N043R-R045T-H249R, G020R-S024R-N043R-R045T-N116A, G020R-S024R-N043R-R045T-N116A-T213A, G020R-S024R-N043R-R045T-S101A-N269R, G020R-S024R-N043R-R045T-S101A-T213A, G020R-S024R-N043R-S242R, G020R-S024R-N076D-H249R, G020R-S024R-N116A, G020R-S024R-N116A-A215F, G020R-S024R-N116A-T213A, G020R-S024R-N116A-T213A-A215F, G020R-S024R-P052N-Q059A-S216F, G020R-S024R-R045T-A230E-S242R, G020R-S024R-R045T-N116A-N269R, G020R-S024R-R045T-N269R, G020R-S024R-S101A-A215F, G020R-S024R-S101A-N116A, G020R-S024R-S101G-S103A-V104I-L217E-A232V-Q245R-H249R, G020R-S024R-S242R-H249R, G020R-S024R-T213A, G020R-S024R-T213A-A215F, G020R-S078R-S101A-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-S078R-S101G-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-A215F-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-G211Q-T213A-A215F-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N116A-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N116A-G211Q-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-S087D-S101G-S103A-V104I-A232V-Q245R, G020R-S101A-N116A-N269R, G020R-S101A-S103A-V104I-A215F-A232V-Q245R, G020R-S101A-S103A-V104I-G118R-A232V-Q245R, G020R-S101A-S103A-V104L-A232V-Q245R, G020R-S101A-S103A-V104V-A232V-Q245R, G020R-S101A-S103S-V104I-A232V-Q245R, G020R-S101A-S103S-V104V-A232V-Q245R, G020R-S101G-I198L-A215F-A232V-Q245R, G020R-S101G-S103A-A232V-Q245R, G020R-S101G-S103A-V104I-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-A232V-Q245R, G020R-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-S101G-S103A-V104I-G211Q-T213A-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-T213A-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-V150L-A232V-Q245R, G020R-S101G-S103A-V104L-A232V-Q245R, G020R-S101G-S103A-V104V-A232V-Q245R, G020R-S101G-S103G-V104I-A232V-Q245R, G020R-S101G-S103S-V104I-A232V-Q245R, G020R-S101G-S103S-V104L-A232V-Q245R, G020R-S101G-V104I-T213A-A215F-A232V-Q245R, G020R-S101S-S103A-V104I-A232V-Q245R, G020R-S101S-S103A-V104V-A232V-Q245R, G020R-S101S-S103G-V104I-A232V-Q245R, G020R-S101S-S103G-V104V-A232V-Q245R, G020R-S101S-S103S-V104I-A232V-Q245R, G020R-S101S-S103S-V104L-A232V-Q245R, G020R-S101S-S103S-V104V-A232V-Q245R, G020R-S103N-S216F-Q236N-N252R, G020R-S144R-N185I-L233C-Q236N, G020R-S212F-H249R, G020R-S242R-N269R, G020R-T022R-E271L, G020R-T022R-N043R, G020R-T022R-N043R-S212F, G020R-T022R-N043R-W241R, G020R-T022R-S024R-S242R, G020R-T022R-S078R-S242R, G020R-T022R-S078R-W241R, G020R-T022R-S212F-W241R, G020R-T022R-S242R, G020R-T022R-W241R, G020R-T022W-S078R-S101A-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-N116S-T213A-A215F-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-T213A-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-G211Q-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-G211Q-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-I198L-G211Q-T213A-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-T038A-N043R-S101A, G020R-T22W-S078R-S101A-S103A-V104I-N116A-N183D-A232V, G020R-T22W-S078R-S101G-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-T22W-S078R-S101G-S103A-V104I-N116A-T213A-A215F-A232V-Q245R, G020R-T22W-S101A-S103A-V104I-A215F-A232V-Q245R, G020R-T22W-S101A-S103A-V104I-A232V-Q245R, G020R-T22W-S101A-S103A-V104I-G211Q-T213A-A232V-Q245R, G020R-T22W-S101G-S103A-V104I-A232V-Q245R, G020R-T22W-S101G-S103A-V104I-G211Q-A232V-Q245R, G020R-T22W-S103A-V104I-A232V-Q245R, G023A-P052N-S144R-Y192W-S216F, G023A-P052N-Y192W-I198L-N252R, G023A-S024R-N117F-S212M-S216F, G023A-S078R-S216F-Q236N-H249R, G023A-Y091F-S101A-I198L-N252R, G023A-Y091F-V121F-Y192W-Q236N, G025R-E089I-N116A-P239S-A270C, G025R-G046R-V121F, G025R-N043A-E089I-N117F, G025R-S103N-R186K-A194F-T224A, G046R-A194F-S212M, G046R-E089I-Y091F-S101A-N116A, G046R-E089I-Y192W-L233C-A270C, G046R-N183D-N238L, G046R-Q059A-S103N-G211Q-S212M, G046R-Q059A-S106G-L217E-H249R, L148I-T213A-N252R, N018R-G020R-N043D, N018R-G020R-N043D-A230E-S242R, N018R-G020R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-N043D-R045T-A230E, N018R-G020R-N043R-N076D-H249R, N018R-G020R-N043R-N076D-S242R-H249R, N018R-G020R-N043R-R045T-S242R, N018R-G020R-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-R045T, N018R-G020R-S024R-N043D-R045T-L233I-S242R, N018R-G020R-S024R-N043R-R045T-N076D-A230E, N018R-G020R-S024R-N076D-S087D-H249R, N018R-G020R-S024R-N076D-V150L-H249R, N018R-G020R-S024R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-N043D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-N043D-S101G-S103A-V104I-A232V-Q245R, N018R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043R-N076D-A230E, N018R-N043R-R045T-H249R, N018R-N043R-R045T-S101G-S103A-V104I-A232V-Q245R, N018R-N043R-R045T-S242R-H249R, N018R-N043R-S101G-S103A-V104I-A232V-Q245R, N018R-N043R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043R-S242R-H249R, N018R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-N076D-S101G-V104I-A232V-Q245R, N018R-Q245R, N018R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-R045T-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-R045T-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N043D-A230E, N018R-S024R-N043D-N076D-H249R, N018R-S024R-N043D-N076D-H249R-N269R, N018R-S024R-N043D-N076D-S078R-H249R, N018R-S024R-N043D-R045T-H249R, N018R-S024R-N043D-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S024R-N043R-N076D-H249R, N018R-S024R-N043R-N076D-H249R-N269R, N018R-S024R-N043R-N076D-S087D-H249R, N018R-S024R-N043R-N076D-V150L-H249R, N018R-S024R-N076D-G211Q-A215F-H249R, N018R-S024R-N076D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-G211Q-T213A-H249R, N018R-S024R-N076D-H249R, N018R-S024R-N076D-H249R-N269R, N018R-S024R-N076D-N116A-A215F-H249R, N018R-S024R-N076D-N116A-G211Q-A215F-H249R, N018R-S024R-N076D-N116A-G211Q-H249R, N018R-S024R-N076D-N116A-T213A-A215F-H249R, N018R-S024R-N076D-S078R-H249R, N018R-S024R-N076D-S078R-S087D-H249R, N018R-S024R-N076D-S078R-V150L-H249R, N018R-S024R-N076D-S087D-H249R-N269R, N018R-S024R-N076D-S087D-S242R-H249R, N018R-S024R-N076D-S087D-V150L-H249R, N018R-S024R-N076D-S101A-A215F-H249R, N018R-S024R-N076D-S101A-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-S101A-I198L-G211Q-A215V-H249R, N018R-S024R-N076D-S101A-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-H249R, N018R-S024R-N076D-S101A-N116A-T213A-H249R, N018R-S024R-N076D-S101G-V104I-A232V-H249R, N018R-S024R-N076D-S242R-H249R, N018R-S024R-N076D-T213A-A215F-H249R, N018R-S024R-N076D-V104I-H249R, N018R-S024R-N076D-V150L-H249R, N018R-S024R-R045T-S242R, N018R-S024R-S101G-V104I, N018R-S024R-S101G-V104I-A232V, N018R-S024R-S242R, N018R-S024R-V104I-H249R, N018R-S024R-V244R, N018R-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-S087D-S101G-S103A-V104I-A232V-Q245R, N018R-S101G-Q245R, N018R-S101G-S103A-H249R, N018R-S101G-S103A-Q245R, N018R-S101G-S103A-V104I-A232V-Q245R, N018R-S101G-S103A-V104I-V150L-A232V-Q245R, N018R-S101G-V104I-A232V-H249R, N018R-S101G-V104I-A232V-Q245R, N018R-S101G-V104I-H249R, N018R-S103A-A232V-H249R, N018R-S103A-V104I-H249R, N018R-T022R-S024R-N043D-N076D-H249R, N018R-T022R-S024R-N043R-N076D-H249R, N018R-T022R-S024R-N076D-H249R, N018R-T022R-S024R-N076D-S087D-H249R, N018R-T022R-S024R-N076D-V150L-H249R, N018R-T022W-S024R-N076D-N116A-G211Q-H249R, N018R-T022W-S024R-N076D-N116A-T213A-H249R, N018R-T022W-S024R-N076D-S101A-I198L-A215F-H249R, N018R-T022W-S024R-N076D-S101A-I198L-H249R, N018R-T022W-S024R-N076D-S101A-N116A-A232V-Q245R, N018R-T22K-N043D-S101G-S103A-V104I-A232V-Q245R, N018R-T22W-S024R-N076D-N116A-T213A-H249R, N018R-T22W-S024R-N076D-S101A-N116A-A232V-Q245R, N018R-V104I-A232V-H249R, N043A-N062Q-A194F-G211Q, N043A-T057R-N117F-S144R-N183D, N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N043D-R045T-N076D-H249R, N043D-S078R-S101G-S103A-V104I-A232V-Q245R, N043D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N043R-N076D-S101G-S103A-V104I-A232V-Q245R, N043R-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-N076D-S242R-H249R, N043R-N116A-N269R, N043R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N043R-R045T-S078R-S101G-S103A-V104I-A232V-Q245R, N043R-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-S078R-S101G-S103A-V104I-A232V-Q245R, N043R-S087D-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-S101A-N116A-A215F-N269R, N043R-S101A-N116A-N269R, N043R-S101A-N116A-T213A-A215F-N269R, N043R-S101A-N269R, N043R-S101A-T213A-N269R, N043R-S101G-S103A-V104I-A232V-Q245R, N043R-S101G-S103A-V104I-A232V-Q245R-E271L, N043R-S101G-S103A-V104I-A232V-Q245R-H249R, N043R-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-S101G-S103A-V104I-A232V-S242R-Q245R, N043R-S101G-S103A-V104I-V150L-A232V-Q245R, N043R-S242R-H249R, N043R-T213A-A215F-N269R, N062Q-S103N-N117F-A194F, N062Q-S103N-V121F-S144R-H249R, N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N076D-S101G-S103A-V104I-A232V-Q245R, N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N076D-S103A-V104I-Q109R, N076D-S103A-V104I-Q109R-Q245R, N076D-S103A-V104I-S212P-E271V, N076D-S103A-V104I-S212P-Q245L-E271V, N076D-S87R-S103A-V104I-S212P-E271V, N117F-A194F-T213A-A270C, N117F-T213A-A215F, P052N-S078R-S103N-L148I-T213A, P052N-S103N-N116A-L148I-Y192W, R019H-G020R-T022W-S078R-S101G-S103A-V104I-G211Q-A232V-Q245R, R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, R045T-S078R-S101G-S103A-V104I-A232V-Q245R, R045T-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S009A-G020R-N043R-S212F, S009A-G020R-N043R-W241R, S009A-G020R-S024R-N043R, S009A-N043R-S078R, S009A-N043R-S078R-S242R, S009A-N043R-S212F, S009A-N043R-W241R, S009A-T022R-N043R-S078R, S009A-T022R-S078R-S212F, S009A-T022R-S078R-S212F-W241R, S009A-T022R-S212F-W241R, S024R-A215F-N269R, S024R-A232V-Q245R, S024R-G025R-N183D-Y192W-P239S, S024R-N043A-N117F-A194F-G211Q, S024R-N043D-H249R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-N043R-A215F, S024R-N043R-A230E, S024R-N043R-A230E-S242R, S024R-N043R-H249R, S024R-N043R-N076D-H249R, S024R-N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-N076D-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-N116A-A215F, S024R-N043R-N116A-A215F-N269R, S024R-N043R-N116A-N269R, S024R-N043R-N116A-T213A-N269R, S024R-N043R-R045T-N076D-A230E-H249R, S024R-N043R-R045T-N269R, S024R-N043R-R045T-S101A-N116A-A215F-N269R, S024R-N043R-R045T-S101A-N116A-T213A-N269R, S024R-N043R-R045T-S242R, S024R-N043R-R045T-T213A-A215F-N269R, S024R-N043R-S101A-A215F, S024R-N043R-S101A-A215F-N269R, S024R-N043R-S101A-N116A, S024R-N043R-S101A-N116A-A215F-N269R, S024R-N043R-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-S242R, S024R-N062Q-V104L-S106G-H249R, S024R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-N076D-V104I-A232V-Q245R, S024R-N116A-T213A-N269R, S024R-R045T, S024R-R045T-N076D-A230E-S242R-H249R, S024R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-S078R-S212F, S024R-S078R-V104L-N116A-N183D, S024R-S087D-S101G-S103A-V104I-A232V-Q245R, S024R-S101A-H120E-A194F-H249R, S024R-S101A-N269R, S024R-S101G-S103A-V104I-A232V-Q245R, S024R-S101G-S103A-V104I-V150L-A232V-Q245R, S024R-S101G-V104I-Q245R, S024R-S103A-Q245R, S024R-S103A-V104I-A232V-H249R, S024R-S103A-V104I-H249R, S024R-S103A-V104I-Q245R, S024R-Y167W-T224A-H249R, S078R-S087D-S101G-S103A-V104I-A232V-Q245R, S078R-S101G-S103A-V104I-A232V-Q245R, S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S078R-S101G-S103A-V104I-V150L-A232V-Q245R, S078R-S103N-S106G-Y167W-Q236N, S078R-V104L-T213A-A215F-T224A, S078R-Y091F-V121F-L233C-N252R, S087D-S101G-S103A-V104I-A232V-Q245R-N269R, S087R-S101G-S103A-V104I-Q109R-S212P-A232V-Q245R-E271V, S099F-S144R-Y167W-N252R, S101A-H120E-Y192W-A215F-T224A, S101A-S103A-V104I-T213A-A232V-Q245R-N269R, S101G-S103A-V104I-A232V-Q245R, S101G-S103A-V104I-A232V-Q245R-E271L, S101G-S103A-V104I-A232V-Q245R-H249R, S101G-S103A-V104I-A232V-Q245R-N269R, S101G-S103A-V104I-A232V-Q245R-N269R, S101G-S103A-V104I-A232V-S242R-Q245R, S101G-S103A-V104I-A232V-V244R-Q245R, S101G-S103A-V104I-G115R-A232V-Q245R, S101G-S103A-V104I-N116A-T213A-A232V-Q245R-N269R, S101G-S103A-V104I-Q109R-A232V-Q245R, S101G-S103A-V104I-Q109R-S212P-A232V-Q245R, S101G-S103A-V104I-Q109R-S212P-A232V-Q245R-E271V, S101G-S103A-V104I-S212F-A232V-Q245R, S101G-S103A-V104I-V150L-A232V-Q245R-H249R, S101G-S103A-V104I-V150L-A232V-Q245R-N269R, S105T-S128N-S144R-L148I-S212M, S106G-N117F-N238L, S144R-G211Q-N238L-P239S-H249R, T022R-N043R-S101G-S103A-V104I-A232V-Q245R, T022R-N076D-S101G-S103A-V104I-A232V-Q245R, T022R-S087D-S101G-S103A-V104I-A232V-Q245R, T022R-S101G-S103A-V104I-A232V-Q245R, T022W-S078R-Y167W-S212M-A270C, T057R-E089I-I198L, T057R-S099F-S105T-I198L-T213A, T057R-S099F-V121F-N185I-Y192W, T057R-Y167W-H249R, V004R-G020R-H249R, V004R-G020R-N043R, V004R-G020R-S024R, V004R-G020R-S024R-H249R, V004R-G020R-S024R-N043R, V004R-G020R-S024R-N043R-S242R, V004R-G020R-S024R-V244R, V004R-N043R-S101G-S103A-V104I-A232V-Q245R, V004R-S009A-G020R-N043R, V004R-S009A-G020R-N043R-S242R, V004R-S009A-G020R-S024R-S242R, V004R-S009A-G020R-S242R, V004R-S009A-N043R-W241R, V004R-S009A-S024R-N043R-W241R, V004R-S009A-S078R-W241R, V004R-S009A-W241R, V004R-S101G-S103A-V104I-A232V-Q245R, V104L-L217E-T224A-H249R-N252R, V104L-T213A-S216F, V121F-N252R-A270C, Y091F-S099F-S101A-S105T-Y167W, N043R-N076D-A230E-H249R, S024R-N043D-N076D-S242R-H249R, G020R-S024R-N043D-R045T, N043R-N076D-H249R, N043R-R045T-H249R, G020R-N043R-N076D, A230E-H249R, N043D-S242R-H249R, G020R-S024R-N043D-N076D-S242R-H249R, S024R-N043D-S242R-H249R, N018R-N043D-R045T-N076D-S242R-H249R, S024R-N043R-N076D, N018R-G020R-H249R, N018R-G020R-N043R, G020R-S024R-T381-N043R-R045T-N076D-S242R-H249R, S024R-N043R-N076D-A230E-S242R, N018R-G020R-N043D-R045T-N076D-S242R, N018R-S024R-N043D-A230E-H249R, N018R-N043R-N076D-S242R-H249R, G020R-S024R-K27R-N043D-S242R-H249R, G020R-S242R-H249R, N018R-N043R-R045T-S242R, G020R-N043R-N076D-A230E-H249R, N018R-N043R-N076D-S242R, N018R-G020R-N043R-N076D, N018R-S024R-N043D-S242R, R045T-S242R-H249R, N018R-G020R-N043R-R045T-N076D-H249R, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
7 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: A016S-S128N-R186H-E271F, N076D-S101G-S103A-V104I-A158E-S188D-M222S-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R-E271G, S024R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, T022A-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R-E271F, T022A-S101G-G102A-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, A016S-A158E-H249R, A016S-A158E-H249R-E271F, A016S-A158E-R186H-H249R, A016S-L111V-S188D-, A016S-T022A-A158E-R186H-E271F, A048R-S078N-N116L-N185E-L217E-P239G, A048R-S128D-N185E-P239G, A158E-R186H-H249R-E271F, E089P-S101A-P129E-R186H, G020K-G023A-N043W-N116L-S188D-T213A-P239G, G020K-G023A-N062E-N116L-G118R-T213A, G020K-G023A-S024F-N062E-S188D-T213A-P239G, G020K-K027R-P129E-S166D-P239G, G020K-S024F-N062E-P239G, G020K-S024F-T033S-P129E-A194E, G023A-G100S-A194E-S212M, G023A-N043W-N116L-G118R-S188D, G023A-S024F-G118R-S188D-P239G, G023A-S024F-K027R-N062E, G023A-S024F-N043W-N062E-N116L-G118R, G023A-S024F-N116L-G118R-S188D-T213A, G023A-S024F-N116L-S188D-P239G, K027R-G100S-G118R-S160D-S188D-N243F, K027R-S101D-S103N-S105T-A272F, N043R-N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R, N062E-S078N-N116L-T224A, N062E-V104L-A158E-R186H-S188D-H249R, N076D-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R-E271F, N076D-S101G-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S024F-G102A-R186K-T213A-L217E-N243F, S024F-N043W-G118R-S188D, S024F-N043W-V104L-V121F-P129E, S024F-N116L-G118R-S188D-P239G, S024F-S103N-V104L-G118R-S188D, S024R-N043R-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-N043R-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, S024R-S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-S128N-A158E-G159E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-S128N-P129E-S188D-A232V-Q245R-N248D, S101A-L111V-P129E, S101A-S103A-V104L-G159E-A232V-Q245R-N248D-H249R, S101A-S103S-V104L-G159E-A232V-Q245R-N248D-H249R, S101A-V104L-S128N-A158E-R186H, S101G-S103A-V104I-P129E-L148I-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-A158E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-S188D-A232V-Q245R-N248D-H249R, S101G-S103S-V104L-G159E-A232V-Q245R-N248D-H249R, S101S-S103A-V104L-G159E-A232V-Q245R-N248D-H249R, S128N-P129E-R186H, T022A-A158E-R186H-H249R-E271F, T022A-S024K-S101G-S103A-V104I-S128N-A158E-G159E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S103A-V104I-S128I-G159D-S188D-A232V-N248D, T022A-S101G-S103A-V104I-L148I-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-G159E-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-P129E-A158E-A232V-N238R-Q245R-N248D, T022A-S128N-A158E-R186H, T022A-S128N-R186H-S188D-, T022A-V104L-A158E-H249R, T022A-V104L-R186H-S188D-H249R, T022L-G023A-K027R-S101D-V104L-S216F, T022L-S078N-N116L-P129E-S256N, T022L-S078N-S128D-T213A, T033S-G118R-P129E-A194E-P239G, T033S-S105T-S188D-S216F, V026F-V104L-S256N-A272F, T022A-N043R-S103A-V104I-S128I-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-N043R-S103A-V104I-G118R-G159D-S188D-L217D-A232V-N248D, T022A-S101D-S103A-V104I-G118R-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, T022A-S103A-V104I-G118R-P129E-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-S103A-V104I-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, T022A-S103A-V104I-S128I-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S024R-S101G-S103A-V104I-P129E-L148I-A158E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-L148I-A158E-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-L148I-G159E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-A158E-A232V-Q245R-N248D-H249R, V104L-L148I-S188D-H249R, S024R-S101A-S103A-V104I-A158E-S166D-S188D-L217E-A232V-Q245R-N248D-H249R, N076D-S101G-S103A-V104I-S128L-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, N043R-N076D-S101G-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, T022A-N043R-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R-E271F, S101A-S103G-V104V-G159E-A232V-Q245R-N248D-H249R, S101S-S103A-V104V-G159E-A232V-Q245R-N248D-H249R, G023A-S024F-V051W-A158E, K027R-T038F-G102A-N116L, N062E-S078N-S144R-S212M, L031F-N116L-S256N-A272F, T022L-T033S-V104L-N116L-S160D-R186K, S024F-G118R-P129E-R186K-T213A, N043W-S105T-T213A-A215D-S216F, L031F-S105T-R186K-S188D, V026F-A194E-T213A-S256N, S103N-S160D-L250I-S256N, S024R-S101G-S103A-V104I-S130A-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-N062E-S103A-V104I-G118R-G159D-S188D-A232V-Q245R-N248D, T022A-N043R-S101D-S103A-V104I-G118R-P129E-G159D-S188D-L217D-A232V, T022A-S024R-S103A-V104I-G159D-S188D-L217D-A232V-N248D, T022A-S024R-N043R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-L217D-A232V-N248D-E271F, T022A-S103A-V104I-G118R-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, G020K-N043W-S188D-T213A, S024F-N062E-G118R-P239G, G023A-N043W-S188D-T213A, G020K-S024F-N043W-N062E-N116L-G118R-S188D-P239G, G020K-N116L-S188D-P239G, G020K-N043W-N062E-G118R, G020K-N043W-N116L-S188D-T213A, G020K-S024F, G023A-N043W-N116L-P239G, G023A-S024F-N043W-N116L-G118R-S188D-P239G, G020K-G023A-N043W-T213A, and G023A-S024F-N062E-G118R-T213A-P239G, and wherein the total net charge of the variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
8 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I-A232V-Q236H-Q245R-N252K, S101G-S103A-V104I-A232V-Q245R-N248R, S101G-S103A-V104I-G159R-A232V-Q245R-N248D, A232V-Q245R, E089I-N117F-N185I-A215F-L233C, G020R-E089I-L217E, G020R-G023A-V104L-Y192W-L233C, G020R-N043D-R045T-N076D-S242R-H249R, G020R-N043D-R045T-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, G020R-N043R-N076D-A230E-H249R, G020R-N043R-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-N116A-A215F-N269R, G020R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, G020R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, G020R-R045T-N116A-N269R, G020R-R045T-S101G-S103A-V104I-A232V-Q245R, G020R-S024R-A215F, G020R-S024R-K27R-N043D-S242R-H249R, G020R-S024R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-S024R-N043R-N076D, G020R-S024R-N043R-R045T-A215F, G020R-S024R-N043R-R045T-N076D-S242R-H249R, G020R-S024R-N043R-R045T-N116A-T213A-A215F, G020R-S024R-N076D-S101G-S103A-V104I-A232V-Q245R, G020R-S024R-T381-N043R-R045T-N076D-S242R-H249R, G020R-S078R-S101A-S103A-V104I-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-G211Q-T213A-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-N116A-N183D-I198L-A215F-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-N183D-A215F-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N116A-A131V-N183D-T213A-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V, G020R-S078R-S101G-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-N183D-G211Q-T213A-A232V-Q245R-E271G, G020R-S078R-S101G-S103A-V104I-T180A-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-S101A-S103A-V104I-T213A-A215F-A232V-Q245R, G020R-S101A-S103G-V104V-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-G211Q-A232V-Q245R, G020R-S101G-S103A-V104I-N183D-G211Q-A232V-Q245R-, G020R-S103A-V104I-A232V-Q245R, G020R-S242R-H249R, G020R-T022W-S078R-S101A-S103A-V104I-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-I198L-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-N116A-N183D-T213A-A215F-A232V-Q245R, G020R-T022W-S078R-S101A-S103A-V104I-N183D-I198L-T213A-A215F-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-G211Q-T213A-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-T022W-S101A-N116A-I198L-G211Q-T213A-A215F-H249R, G020R-T022W-S101A-S103A-V104I-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-G211Q-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-G211Q-T213A-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-I198L-G211Q-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-G211Q-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-G211Q-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-A114T-T213A-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-G211Q-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-I198L-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-T213A-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N183D-I198L-T213A-A215F-A232V-Q245R, G020R-T022W-S103A-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S103A-V104I-A232V-Q245R, G020R-T22W-S078R-S101A-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-T22W-S078R-S101A-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-T22W-S101A-S103A-V104I-G211Q-A215F-A232V-Q245R, G020R-T22W-S101A-S103A-V104I-N116A-G211Q-A215F-A232V-Q245R, G020R-T22W-S101A-S103A-V104I-N116A-G211Q-T213A-A215F-A232V-Q245R, G020R-T22W-S101G-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-T22W-S101G-S103A-V104I-N116A-N183D-T213A-A215F-A232V-Q245R, G025R-N043A-Y091F-I198L-A270C, G025R-S105T-S128N-S144R-A270C, G046R-E089I-S099F-R186K-S212M, G118R-A172V, G118R-A194T, G118R-Y209A-K235F, H017Y-S024R-T033S-P086W, I008T-S024R, K027R-N043R-S101G-S103A-V104I-A232V-Q245R-N269R, K027R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, K235F-N243F, N018R-G020R-H249R, N018R-G020R-N043D-H249R, N018R-G020R-N043D-R045T-A230E-S242R, N018R-G020R-N043D-R045T-N076D-S242R, N018R-G020R-N043D-R045T-S240P, N018R-G020R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-N043D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-N043R, N018R-G020R-N043R-N076D, N018R-G020R-N043R-N076D-A230E-S242R, N018R-G020R-N043R-R045T-N076D-H249R, N018R-G020R-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-R045T-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-S024R-N043D-N076D-S242R, N018R-G020R-S024R-N043D-R045T-S242R, N018R-G020R-S024R-N043R-N076D-H249R, N018R-G020R-S024R-N043R-R045T-N076D-H249R, N018R-G020R-S024R-N076D-A215F-H249R, N018R-G020R-S024R-N076D-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-L217E-H249R, N018R-G020R-S024R-N076D-N116A-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N116A-N183D-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N183D-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-I198L-H249R, N018R-G020R-S024R-N076D-S101A-N183D-G211Q-A215F-H249R, N018R-G020R-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-T213A-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-H249R, N018R-G020R-T22W-S024R-N076D-N116A-N183D-I198L-T213A-H249R, N018R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N043D-R045T-N076D-S242R-H249R, N018R-N043D-R045T-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-N043D-R045T-S101G-S103A-V104I-A232V-Q245R, N018R-N043D-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N043R-N076D-A230E-S242R-H249R, N018R-N043R-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043R-N076D-S242R-H249R, N018R-N043R-R045T-N076D-A230E-S242R, N018R-N043R-R045T-N076D-S076T-S101G-S103A-V104I-A232V-Q245R-A273T, N018R-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043R-R045T-N076D-S242R, N018R-N043R-R045T-S242R, N018R-N076D-H249R, N018R-N076D-Q245R, N018R-N076D-S101G-A232V-Q245R, N018R-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N076D-S101G-S103A-V104I-H249R, N018R-N076D-S101G-V104I-H249R, N018R-N076D-S103A-V104I-H249R, N018R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N043D-A230E-H249R, N018R-S024R-N043D-A230E-S242R, N018R-S024R-N043D-H249R, N018R-S024R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N043D-N076D-S242R-H249R, N018R-S024R-N043D-N076D-V150L-H249R, N018R-S024R-N043D-S242R, N018R-S024R-N043D-S242R-H249R, N018R-S024R-N043R-N076D-L217E-H249R, N018R-S024R-N043R-N076D-S078R-H249R, N018R-S024R-N043R-R045T-A230E-H249R, N018R-S024R-N076D, N018R-S024R-N076D-A232V-H249R, N018R-S024R-N076D-G211Q-H249R, N018R-S024R-N076D-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-I198L-T213A-A215F-H249R, N018R-S024R-N076D-I198M-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-L088I-S101A-N116A-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-N116A-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N116A-H249R, N018R-S024R-N076D-N116A-I198L-A215F-H249R, N018R-S024R-N076D-N116A-I198L-A215F-H249R-V268G, N018R-S024R-N076D-N116A-I198L-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N116A-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-N116A-I198L-H249R, N018R-S024R-N076D-N116A-I198L-T213A-H249R, N018R-S024R-N076D-N116A-I198L-Y209H-T213A-H249R, N018R-S024R-N076D-N116A-M117I-N183D-T213A-H249R, N018R-S024R-N076D-N116A-N183D-G211Q-H249R, N018R-S024R-N076D-N116A-T213A-H249R, N018R-S024R-N076D-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N183D-T213A-A215F-H249R, N018R-S024R-N076D-S101A-G211Q-A215F-H249R, N018R-S024R-N076D-S101A-G211Q-H249R, N018R-S024R-N076D-S101A-H249R, N018R-S024R-N076D-S101A-I198L-A215F-H249R, N018R-S024R-N076D-S101A-I198L-G211Q-H249R, N018R-S024R-N076D-S101A-I198L-H249R, N018R-S024R-N076D-S101A-I198L-T213A-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-I198L-G211Q-H249R, N018R-S024R-N076D-S101A-N116A-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-I198L-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-T213A-H249R, N018R-S024R-N076D-S101A-N116A-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N183D-G211Q-H249R, N018R-S024R-N076D-S101A-N183D-T213A-H249R, N018R-S024R-N076D-S101G-A232V, N018R-S024R-N076D-S103A-A232V-Q245R, N018R-S024R-N076D-T213A-H249R, N018R-S024R-N076D-V104I, N018R-S024R-Q245R, N018R-S024R-R045T-A230E-S242R, N018R-S024R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-S024R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S024R-S242R, N018R-S024R-V104I, N018R-S024R-V30S-L31S-D32I-T33Q-G34V-I35F, N018R-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-S101G-S103A-V104I, N018R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S101G-V104I, N018R-S103A, N018R-S103A-V104I-A232V, N018R-T022K-N043D-S101G-S103A-V104I-A232V-Q245R, N018R-T022W-S024R-N076D-G211Q-H249R, N018R-T022W-S024R-N076D-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-I198L-A215F-H249R, N018R-T022W-S024R-N076D-I198L-G211Q-H249R, N018R-T022W-S024R-N076D-I198L-T213A-A215F-H249R, N018R-T022W-S024R-N076D-N116A-G211Q-A215F-H249R, N018R-T022W-S024R-N076D-N116A-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-N116A-I198L-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-N116A-I198L-T213A-H249R, N018R-T022W-S024R-N076D-N116A-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-G211Q-H249R, N018R-T022W-S024R-N076D-S101A-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-S101A-I198L-G211Q-Q245R, N018R-T022W-S024R-N076D-S101A-I198L-G211Q-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-I198L-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N116A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N116A-G211Q-H249R, N018R-T022W-S024R-N076D-S101A-N116A-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-G211Q-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-G211Q-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N116A-I198L-T213A-H249R, N018R-T022W-S024R-N076D-S101A-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-T213A-H249R, N018R-T022W-S024R-N076D-T213A-A215F-H249R, N018R-T22W-S024R-N076D-I198L-A215F-H249R, N018R-T22W-S024R-N076D-N116A-G211Q-H249R, N018R-T22W-S024R-N076D-N116A-G211Q-T213A-H249R, N018R-T22W-S024R-N076D-N116A-T213A-A215F-H249R, N018R-T22W-S024R-N076D-S101A-A215F-H249R, N018R-T22W-S024R-N076D-S101A-G211Q-T213A-H249R, N018R-T22W-S024R-N076D-S101A-I198L-A215F-H249R, N018R-T22W-S024R-N076D-S101A-I198L-T213A-A215F-H249R, N018R-T22W-S024R-N076D-S101A-N116A-A215F-H249R, N018R-T22W-S024R-N076D-S101A-N116A-I198L-A215F-H249R, N018R-T22W-S024R-N076D-T213A-A215F-H249R, N018R-V104I, N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N043D-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N043D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R-A272V, N043D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R-R275S, N043D-S101G-S103A-V104I-A232V-Q245R, N043D-S101G-S103A-V104I-A232V-Q245R-H249R, N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N043R-N076D-S101G-S103A-V104I-L217E-A232V-Q245R, N043R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-V234I-Q245R, N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-R045T-S078R-S101G-S103A-V104I-N218S-A232V-Q245R, N043R-R045T-S101G-S103A-V104I-A232V-Q245R, N043R-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-S087D-S101G-S103A-V104I-A232V-Q245R, N062Q-S101A-Q236N-N252R-A270C, N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N076D-S101G-S103A-V104I-A232V-S242R-Q245R, P005S-S024R-T033S-N243F, P014L-G020R-S024R-N043R-R045T-S101A-A215F, P052N-V104L-N183D-S216F-H249R, P086W-G118R, P086W-N243F, P086W-Y209A, Q012H-G020R-S078R-S101G-S103A-V104I-I198L-G211Q-T213A-A232V-Q245R, R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, R045T-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, R045T-S101G-S103A-V104I-A232V-Q245R-N269R, R045T-S242R-H249R, S024C-T033S, S024R-A232V, S024R-G046R-Y091F-V121F, S024R-G118R-K235F, S024R-N043A-S105T-S106G-I198L, S024R-N043D-R045T-S242R-H249R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R, S024R-N043D-S242R-H249R, S024R-N043R-N076D, S024R-N043R-N076D-A230E-S242R, S024R-N043R-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-N043R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-R045T-S242R-H249R, S024R-N043R-S101A-T213A, S024R-N076D-Q245R, S024R-N076D-S101G-A232V-Q245R, S024R-N076D-S101G-M175L-A232V-Q245R, S024R-N076D-S101G-Q245R, S024R-N076D-S101G-S103A-V104I-A232V-Q245R, S024R-N076D-S103A-V104I-Q245R, S024R-N076D-V104I-Q245R, S024R-N243F, S024R-P086R-G118R, S024R-P086W-Y209A-K235F, S024R-P239Q, S024R-Q245R-H249R, S024R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-S101G, S024R-S101G-Q245R, S024R-S101G-V104I-H249R, S024R-S103A-A232V, S024R-S103A-M175L-A232V-H249R, S024R-S141G, S024R-T033S, S024R-T033S-A194T, S024R-T033S-G118R-K235F, S024R-T033S-G118R-Y209A, S024R-T033S-K235F, S024R-T033S-S078R-G118R, S024R-T033S-S078R-N243F, S024R-T033S-Y209A, S024R-T033S-Y209A-N243F, S024R-T274I, S024R-V104I-H249R, S024R-V104I-Q245R, S024R-Y091F-I198L-A215F-P239S, S024R-Y209A, S024R-Y209A-K235F, S024R-Y209A-S242P, S078R-P086W, S078R-S099F-N116A-R186K-T224A, S078R-S101G-S103A-V104I-A232V-Q245R-H249R, S101G-A232V, S101G-S103A-V104I-A232V-H249R, S101G-S103A-V104I-A232V-Q245R-H249R-N269R, S101G-S103A-V104I-Q245R, S105T-G211Q-S216F, S106G-N185I-S216F-Q236N, T022R-S101G-S103A-V104I-V150L-A232V-Q245R, T033S-G118D-A138V-Y209A, T033S-G118R-Y209A-, T033S-L148F, T033S-N243F, T033S-P086W, T033S-P201S, T033S-S078R, T033S-W241R, T033S-Y209A, T033S-Y209A-K235F, V004M-N018R-S024R-N076D-N116A-I198L-G211Q-T213A-H249R, V104I-A232V-H249R, and V104L-H120E-R186K-S216F-N252R, and wherein the total net charge of the protease variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
9 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: A015T-T033S, A016T-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, A230E-H249R, A232V-H249R, E089I-S105T-N116A-A215F-S216F, E089I-Y091F-N185I-G211Q-A270C, G020R-A090S-S101G-S103A-V104I-N116A-N183D-T213A-A215F-A232V-Q245R, G020R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, G020R-N043R-N076D, G020R-N043R-R045T-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-P052N-S101A-I198L-L233C, G020R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-S024R-N043D-N076D-H249R, G020R-S024R-N043D-N076D-S242R-H249R, G020R-S024R-N043D-R045T-A230E-S242R, G020R-S024R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-S024R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, G020R-S024R-N043R-S101G-S103A-V104I-A232V-Q245R, G020R-S024R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, G020R-S024R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, G020R-S024R-R045T-N076D-S242R-H249R, G020R-S024R-R045T-S101G-S103A-V104I-A232V-Q245R, G020R-S078R-S101A-S103A-V104I-G115E-N116A-N183D-G211Q-T213A-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R-T274I, G020R-S101A-S103A-V104I-N116A-N183D-I198L-G211Q-T213A-A215F-A232V-Q245R, G020R-S101A-S103A-V104I-N116A-N183D-T213A-A215F-A232V-Q245R, G020R-S101A-S103A-V104I-T213A-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-N183D-G211Q-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-N183D-G211Q-T213A-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-T022R-N269R, G020R-T022R-S078R-S212F-W241R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-A232V-Q245R-N269S, G020R-T022W-S101A-S103A-V104I-N116A-N183D-A232V-Q245R-T274I, G020R-T022W-S101A-S103A-V104I-N116A-N183D-G211Q-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-I198L-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-T213A-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N183D-I198L-A215F-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N183D-T213A-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-G211Q-A232V-Q245R-N263S, G020R-T022W-S101G-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-G211Q-T213A-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N183D-A215F-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-N183D-A232V-Q245R, G025R-N062Q-S128N-S144R-N185I, G025R-N116A-H120E-T224A-A270C, G118R-K235F, K027R-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018K-N076D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R-N269R, N018R-G020R-N043D-N076D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-G020R-N043D-N076D-S242R-H249R, N018R-G020R-N043D-R045T-N076D-H249R, N018R-G020R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-N043D-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-G020R-N043R-R045T-H249R, N018R-G020R-N043R-R045T-N076D-A230E-H249R, N018R-G020R-N076D, N018R-G020R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-R045T-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-S024R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-S024R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N043R-N076D, N018R-G020R-S024R-N076D-A131T-A215F-H249R, N018R-G020R-S024R-N076D-G211Q-H249R, N018R-G020R-S024R-N076D-G211Q-N243D-H249R, N018R-G020R-S024R-N076D-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-I198L-A215F-H249R, N018R-G020R-S024R-N076D-I198L-H249R, N018R-G020R-S024R-N076D-I198L-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N116A-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N116A-H249R, N018R-G020R-S024R-N076D-N116A-I198L-G211Q-A215F-H249R-N269S, N018R-G020R-S024R-N076D-N116A-I198L-G211Q-A215F-Q245R, N018R-G020R-S024R-N076D-N116A-I198L-H249R, N018R-G020R-S024R-N076D-N116A-N183D-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-N116A-N183D-I198L-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-N116A-N183D-I198L-H249R, N018R-G020R-S024R-N076D-N116A-N183D-T213A-H249R, N018R-G020R-S024R-N076D-N183D-A215F-H249R, N018R-G020R-S024R-N076D-N183D-G211Q-H249R, N018R-G020R-S024R-N076D-N183D-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N183D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-N183D-I198L-A215F-H249R, N018R-G020R-S024R-N076D-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N204D-T213A-H249R, N018R-G020R-S024R-N076D-S101A-G211Q-A215F-H249R-N269D, N018R-G020R-S024R-N076D-S101A-H249R, N018R-G020R-S024R-N076D-S101A-I198L-T213A-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N116A-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N116A-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-I198L-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N116A-T213A-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N116A-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N183D-G211Q-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N183D-T213A-A215F-H249R, N018R-G020R-S024R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-S024R-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-I198L-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-I198L-H249R, N018R-G020R-T022W-S024R-N076D-N116A-I198L-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N183D-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N183D-I198L-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-S101A-I198L-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-T213A-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-T213A-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-T213A-H249R, N018R-N043D-A230E-H249R, N018R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N043D-N076D-S242R-H249R, N018R-N043D-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-N043D-R045T-S078R-S101G-S103A-V104I-A232V-Q245R-A272D, N018R-N043R-N076D-A230E-H249R, N018R-N043R-N076D-S242R, N018R-N043R-R045T-A230E-S242R, N018R-N043R-R045T-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-N043R-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-N043R-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N076D-A232V-H249R, N018R-N076D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-N076D-S101G-I198T-A232V, N018R-N076D-S101G-S103A-V104I-Q245R, N018R-N076D-S101G-V104I, N018R-N076D-S242R, N018R-N076D-S242R-H249R, N018R-N076D-V104I-Q245R-H249R, N018R-R045T-H249R, N018R-R045T-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S024R-N043D-N076D-S087D-H249R, N018R-S024R-N043D-N076D-S242R, N018R-S024R-N076D-A086V-S101A-N183D-I198L-G211Q-H249R, N018R-S024R-N076D-A215F-H249R, N018R-S024R-N076D-A232V, N018R-S024R-N076D-I198L-A215F-H249R, N018R-S024R-N076D-I198L-G211Q-H249R, N018R-S024R-N076D-I198L-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-I198L-H249R, N018R-S024R-N076D-I198L-T213A-H249R, N018R-S024R-N076D-L217E-H249R-N269R, N018R-S024R-N076D-N116A-A150T-T213A-H249R, N018R-S024R-N076D-N116A-G211Q-T213A-H249R, N018R-S024R-N076D-N116A-I198L-G211Q-H249R, N018R-S024R-N076D-N116A-I198L-T213A-A215F-H249R, N018R-S024R-N076D-N116A-N183D-A215F-H249R, N018R-S024R-N076D-N116A-N183D-G211Q-A215F-H249R, N018R-S024R-N076D-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N116A-N183D-G211Q-T213A-H249R, N018R-S024R-N076D-N116A-N183D-H249R, N018R-S024R-N076D-N116A-N183D-I198L-A215F-H249R, N018R-S024R-N076D-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-N116A-N183D-I198L-G211Q-H249R, N018R-S024R-N076D-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-N116A-N183D-I198L-H249R, N018R-S024R-N076D-N116A-N183D-I198L-T213A-H249R, N018R-S024R-N076D-N116A-N183D-T213A-A215F-H249R, N018R-S024R-N076D-N116A-N183D-T213A-H249R, N018R-S024R-N076D-N183D-A215F-H249R, N018R-S024R-N076D-N183D-G211Q-H249R, N018R-S024R-N076D-N183D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N183D-G211Q-T213A-H249R, N018R-S024R-N076D-N183D-H249R, N018R-S024R-N076D-N183D-H249R-A248T, N018R-S024R-N076D-N183D-I198L-A215F-H249R, N018R-S024R-N076D-N183D-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-N183D-I198L-G211Q-H249R, N018R-S024R-N076D-N183D-I198L-H249R, N018R-S024R-N076D-N183D-I198L-T213A-A215F-H249R, N018R-S024R-N076D-N183D-I198L-T213A-H249R, N018R-S024R-N076D-N183D-T213A-H249R, N018R-S024R-N076D-S087D-H249R, N018R-S024R-N076D-S101A-G211Q-T213A-A215F-H249R-A270V, N018R-S024R-N076D-S101A-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-A215F-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-H249R, N018R-S024R-N076D-S101A-N116A-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-S101A-N116A-I198L-H249R, N018R-S024R-N076D-S101A-N116A-I198L-T213A-H249R, N018R-S024R-N076D-S101A-N116A-N183D-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-G211Q-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-G211Q-H249R, N018R-S024R-N076D-S101A-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-N183D-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-T213A-A215F-H249R, 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G020R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N043D-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-H249R, N018R-N076D-V104I, N018R-G020R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-T022W-S024R-N076D-N183D-G211Q-H249R, N018R-S024R-N076D-S101A-N183D-I198L-G211Q-A215F-H249R, S101G-V104I, N018R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N183D-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-A215F-H249R-N263D, N018R-T022W-S024R-N076D-S101A-N183D-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-A215F-H249R, N018R-T022W-S024R-N076D-N116A-I198L-A215F-H249R-R269H, N018R-N043D-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-I198L-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N183D-I198L-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-I198L-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-H249R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-I198L-G211Q-T213A-A215F-A232V-Q245R, V104I-A232V, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N183D-I198L-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N183D-I198L-H249R, N043D-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-G211Q-H249R, N018R-G020R-N043R-S101G-S103A-V104I-L217E-A232V-Q245R, S024R-N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-R045T-N076D-S078R-S101G-S103A-V104I-Y166F-A176P-A179V-N184T-A187P-A194P, N018R-T022W-S024R-D041E-N076D-S101A-S160T-N183D-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-I198L-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N183D-G211Q-A215F-H249R, S024R-N076D-V104I, N018R-N076D-S101G-A232V, N018R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-A215F-H249R, N018R-S024R-L031F-N076D-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-T213A-H249R-N269S, N018R-G020R-S024R-N076D-S101A-N116A-I198L-A215F-H249R, N018R-N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N076D-S101A-N114T-I198L-G211Q-A215F-H249R, S024R-N043R-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, S024R-N076D-S103A-V104I-A232V, N018R-G020R-T022W-S024R-N076D-S101A-I198L-G211Q-A215F-H249R, S101G-V104I-A232V, N018R-T022W-S024R-N076D-N183D-I198L-T213A-A215F-H249R, N018R-T022W-S024R-N076D-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N183D-T213A-H249R, N018R-S024R-N076D-S101A-N183D-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-G211Q-T213A-H249R, N018R-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-G211Q-A215F-H249R-T260A, N076D-H249R, N018R-T022W-S024R-N076D-N183D-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N116A-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-H249R, N076D-V104I-H249R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, S024R-N043R-R045T-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-N183D-I198L-A215F-H249R, N018R-N076D-S101G-S103A, G020R-S024R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-G211Q-H249R, S101G-S103A-A232V, S024R-N076D-S101G-A232V, N018R-T022W-S024R-N076D-S101A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N183D-I198L-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N116A-A156V-N183D-G211Q-A215F-H249R-N269S, R045T-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-H249R, N018R-G020R-T022W-S024R-N076D-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-H249R, N018R-N076D-S101G, N018R-S024R-N076D-N116A-N183D-I198L-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-H249R, N018R-N076D-A232V, N018R-G020R-S024R-N076D-S101A-A232V-Q245R, N018R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-T022W-S024R-N076D-N183D-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-T213A-N237D-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-G211Q-H249R-R275S, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, S024R-N076D, N018R-S024R-N076D-N183D-G211Q-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-I198L-T213A-A215F-H249R, N076D-V104I-A232V-H249R, N018R-N076D-S103A-A232V, N018R-G020R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N183D-T213A-H249R, N018R-G020R-S024R-N076D-S101A-D175E-N183D-G211Q-A215F-H249R, N018R-G020R-N043D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R-A273E, G020R-S024R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, P005S-S101G-S103A-V104I-A232V-Q245R-H249R, S103A-V104I-A232V, N018R-G020R-S024R-V068A-N076D-S101A-N116A-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-I198L-A215F-H249R-R275S, N018R-S024R-N076D-N183D-I198L-G211Q-T213A-H249R, N043D-R045T-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N076D-L217E-S242R-H249R, N076D-S087D-S101G-S103A-V104I-A232V-Q245R, S087D-S101G-S103A-V104I-A232V-Q245R, and S101G-S103A-V104I-V150L-A232V-S242R-Q245R, and wherein the total net charge of the protease variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, and wherein the total net charge is obtained by one or more substitutions selected from: N43D, R45T, N62E, N76D, S101D, P129E, A158E, G159D, G159E, S166D, S188D, A230E, N18R, G20K, G20R, T22R, S24R, N43R, G118R, Q245R, H249R, N269R, E271F, and E271L, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1.
10 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020R-X-43R, X020K-X062E, X024F-X116L, X020K-X024F, X024R-X174T, X024R-X118R, X024R-X235F, X024R-X086R, X024R-X086W, X078R-X118R, X033S-X118R, X033S-X235F, X209A-X241R, X020R-X076D, X018R-X245R, X024R-X045T, X232V-X245R, X118R-X172V, X118R-X194T, X008T-X024R, X235F-X243F, X018R-X103A, X018R-X104I, X086W-X118R, X086W-X243F, X086W-X209A, X024C-X033S, X024R-X232V, X024R-X243F, X024R-X239Q, X024R-X101G, X024R-X141G, X024R-X033S, X024R-X274I, X024R-X209A, X078R-X086W, X101G-X232V, X033S-X148F, X033S-X086W, X033S-X201S, X033S-X078R, X033S-X241R, X033S-X209A, X230E-X249R, X232V-X249R, X118R-X235F, X076D-X245R, X086W-X235F, X024R-X247H, X024R-X104A, X078R-X235F, X101G-X249R, X103A-X232V, X033S-X048T, X033S-X239T, X033S-X253A, X143A-X209A, X209A-X235F, X018R-X045T, X209A-X243F, X024R-X272P, X024R-X269C, X101G-X104I, X104I-X232V, X076D-X249R, and X024R-X076D wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
11 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020R-X076D, X024R-X045T, X230E-X249R, X018R-X045T, X018R-X245R, X101G-X232V, X024R-X232V, X232V-X245R, X024R-X101G, X018R-X104I, X018R-X103A, X101G-X249R, X232V-X249R, X103A-X232V, X076D-X245R, X101G-X104I, X104I-X232V, X076D-X249R, X024R-X076D, X024F-X116L, X020K-X024F, X020K-X062E, X033S-X118R, X024R-X086W, X024R-X118R, X024R-X086R, X209A-X241R, X024R-X241R, X024R-X235F, X118R-X209A, X078R-X118R, X033S-X235F, X024R-X174T, X086W-X209A, X008T-X024R, X086W-X118R, X033S-X241R, X024R-X243F, X024R-X209A, X033S-X086W, X024R-X033S, X086W-X243F, X033S-X201S, X024R-X239Q, X078R-X086W, X235F-X243F, X118R-X172V, X033S-X148F, X033S-X078R, X033S-X243F, X024C-X033S, X118R-X194T, X033S-X209A, X024R-X141G, X024R-X274I, X086W-X235F, X015T-X033S, X209A-X235F, X024R-X247H, X078R-X235F, X024R-X104A, X033S-X048T, X118R-X235F, X033S-X253A, X143A-X209A, X033S-X239T, X209A-X243F, X024R-X272P, and X024R-X269C, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
12 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X004R-X009A-X020R-X242R, X020R-X043R-X241R, X020R-X242R-X269R, X004R-X009A-X020R-X043R, X004R-X020R-X249R, X018R-X024R-X244R, X009A-X022R-X212F-X241R, X020R-X043R-X269R, X018R-X024R-X242R, X004R-X009A-X043R-X241R, X020R-X043R-X244R, X020R-X022R-X242R, X004R-X020R-X043R, X004R-X009A-X020R-X043R-X242R, X020R-X043R-X242R, X020R-X043R-X242R-X249R, X020R-X212F-X249R, X004R-X009A-X241R, X001R-X009A-X043R, X020R-X043R-X249R, X009A-X020R-X043R-X241R, X020R-X022R-X043R, X020R-X249R-X269R, X020R-X022R-X241R, X004R-X009A-X024R-X043R-X241R, X009A-X043R-X078R, X004R-X020R-X024R-X244R, X020R-X022R-X078R-X242R, X020R-X024R-X242R-X249R, X004R-X009A-X078R-X241R, X009A-X043R-X078R-X242R, X004R-X020R-X024R, X009A-X043R-X212F, X020R-X043R-X212F, X024R-X078R-X212F, X009A-X020R-X024R-X043R, X009A-X022R-X043R-X078R, X020R-X022R-X212F-X241R, X020R-X043R-X212F-X241R, X009A-X043R-X241R, X020R-X043R-X271L, X020R-X022R-X078R-X241R, X020R-X024R-X043R-X242R, X020R-X022R-X043R-X241R, X009A-X020R-X043R-X212F, X004R-X009A-X020R-X024R-X242R, X020R-X043R-X249R-X271L, X020R-X022R-X024R-X242R, X009A-X022R-X078R-X212F, X020R-X043R-X242R-X271L, X009A-X022R-X078R-X212F-X241R, X004R-X020R-X024R-X249R, X020R-X022R-X271L, X020R-X022R-X043R-X212F, X004R-X020R-X024R-X043R-X242R, X004R-X020R-X024R-X043R, X004R-X009A-X022R-X078R-X212F, X020R-X022R-X078R-X212F-X241R, and X020R-X022R-X269R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
13 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X018R-X020R-X043D-X045T-X230E, X018R-X043R-X045T-X242R-X249R, X024R-X043D-X249R, X018R-X020R-X045T, X020R-X024R-X076D-X249R, X024R-X043R-X230E-X242R, X018R-X024R-X043D-X230E, X020R-X076D, X018R-X024R-X043D-X076D-X249R, X024R-X043R-X076D-X249R, X018R-X024R-X045T-X242R, X020R-X043D-X076D-X230E-X249R, X020R-X043R-X045T-X242R, X018R-X024R-X076D-X249R, X018R-X020R-X024R-X043D-X045T-X233I-X242R, X024R-X043R-X230E, X018R-X020R-X043D, X043R-X242R-X249R, X020R-X043R-X045T-X230E, X043R-X076D-X242R-X249R, X020R-X024R-X045T-X230E-X242R, X024R-X045T-X076D-X230E-X242R-X249R, X024R-X045T, X024R-X043R-X045T-X076D-X230E-X249R, X018R-X024R-X043D-X045T-X249R, X018R-X043R-X045T-X249R, X024R-X043R-X242R, X018R-X020R-X043R-X076D-X249R, X020R-X024R-X043D-X249R, X020R-X043R-X230E-X242R, X020R-X043R-X242R, X018R-X043R-X076D-X230E, X020R-X024R-X043D-X242R, X020R-X043R-X230E, X018R-X020R-X043R-X076D-X242R-X249R, X043D-X045T-X076D-X249R, X018R-X043R-X242R-X249R, X018R-X020R-X043R-X045T-X242R, X018R-X020R-X043D-X230E-X242R, X020R-X024R-X043R-X045T-X249R, X024R-X043R-X249R, X020R-X024R-X27E-X043R-X076D-X230E, X024R-X043R-X045T-X242R, X018R-X020R-X024R-X043R-X045T-X076D-X230E, X020R-X043R-X076D-X230E-X249R, X018R-X043R-X045T-X242R, X020R-X242R-X249R, X018R-X043R-X076D-X230E-X242R-X249R, X018R-X024R-X076D, X020R-X024R-X27R-X043D-X242R-X249R, X018R-X020R-X024R-X043D-X076D-X242R, X018R-X043R-X076D-X242R-X249R, X018R-X024R-X043D-X230E-X249R, X018R-X020R-X043D-X249R, X018R-X020R-X043D-X045T-X076D-X242R, X024R-X043R-X076D-X230E-X242R, X020R-X024R-X381-X043R-X045T-X076D-X242R-X249R, X018R-X020R-X043R, X018R-X024R-X045T-X230E-X242R, X018R-X020R-X249R, X024R-X043R-X076D, X018R-X020R-X024R-X043R-X045T-X076D-X249R, X018R-X043D-X045T-X076D-X242R-X249R, X024R-X043D-X242R-X249R, X018R-X020R-X024R-X043D-X045T-X242R, X020R-X024R-X043R-X076D, X018R-X020R-X043D-X045T-X230E-X242R, X020R-X024R-X043R-X045T-X076D-X242R-X249R, X018R-X043R-X045T-X076D-X242R, X018R-X020R-X043R-X076D-X230E-X242R, X018R-X024R-X043D-X249R, X018R-X024R-X043R-X045T-X230E-X249R, X018R-X020R-X043R-X045T-X076D-X249R, X018R-X024R-X242R, X018R-X043R-X045T-X076D-X230E-X242R, X045T-X242R-X249R, X018R-X024R-X043D-X242R, X018R-X020R-X043D-X045T-X240P, X024R-X043R-X045T-X242R-X249R, X018R-X024R-X30S-X31S-X32I-X33Q-X34V-X3 X018R-X020R-X043R-X076D, X020R-X043D-X045T-X076D-X242R-X249R, X018R-X024R-X043D-X230E-X242R, X018R-X024R-X043D-X242R-X249R, X024R-X043D-X045T-X242R-X249R, X043R-X230E-X249R, X024R-X043R-X076D-X230E-X249R, X020R-X024R-X043D-X076D-X249R, X024R-X045T-X242R-X273V, X020R-X024R-X045T-X076D-X242R-X249R, X018R-X024R-X043D-X076D-X242R, X018R-X043R-X076D-X230E-X249R, X018R-X020R-X043R-X045T-X249R, X018R-X043R-X045T-X230E-X242R, X020R-X024R-X043D-X045T-X230E-X242R, X018R-X043D-X230E-X249R, X018R-X043R-X076D-X242R, X018R-X020R-X076D, X018R-X020R-X043D-X076D-X242R-X249R, X020R-X024R-X043D-X076D-X242R-X249R, X043D-X242R-X249R, X018R-X020R-X024R-X043R-X076D, X018R-X020R-X043D-X045T-X076D-X249R, X018R-X020R-X043R-X045T-X076D-X230E-X249R, X018R-X076D-X242R, X020R-X043R-X249R, X018R-X076D-X242R-X249R, X018R-X024R-X045T-X230E-X249R, X230E-X249R, X018R-X045T-X249R, X020R-X043R-X076D, X043R-X045T-X249R, X018R-X043D-X076D-X242R-X249R, X043R-X076D-X249R, X018R-X045T, X020R-X076D-X230E-X242R, X020R-X024R-X043D-X045T, X024R-X043D-X076D-X242R-X249R, X020R-X045T-X249R, X043R-X076D-X153A-X249R, X043R-X076D-X230E-X249R, X018R-X043D-X076D-X249R, and X020R-X043R-X076D-X227I, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
14 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101G-X103A-X104I-X232V-X236H-X245R-X252K, X101G-X103A-X104I-X232V-X245R-X248R, X101G-X103A-X104I-X159R-X232V-X245R-X248D, X101G-X103A-X104I-X159D-X232V-X245R-X248R, X101G-X103A-X104I-X232V-X245R, X101G-X103A-X104I-X159D-X232V-X245R, and X101G-X103A-X104I-X232V-X245R-X248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
15 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101G-X103A-X104I-X129E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X158E-X188D-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X129E-X158E-X188D-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X159E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128N-X129E-X158E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X148I-X158E-X188D-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X159E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128N-X129E-X232V-X245R-X248D, X101G-X103A-X104I-X129E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X158E-X159E-X188D-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X159E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X148I-X158E-X232V-X245R-X248D, X016S-X024R-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X158E-X159E-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X129E-X158E-X159E-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X159E-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X128N-X158E-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X188D-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X158E-X188D-X232V-X238R-X245R-X248D, X022A-X101G-X103A-X104I-X128N-X129E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X158E-X159E-X188D-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X128N-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X158E-X188D-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X128N-X129E-X158E-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X159E-X188D-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X128N-X129E-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X159E-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X188D-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X158E-X159E-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X159E-X188D-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X129E-X158E-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X148I-X158E-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X158E-X159E-X188D-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X129E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X158E-X159E-X188D-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X158E-X159E-X188D-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X129E-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128N-X158E-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X128N-X129E-X188D-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X129E-X159E-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X129E-X159E-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X148I-X158E-X188D-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X129E-X159E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X159E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X158E-X159E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X158E-X159E-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X128N-X159E-X188D-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X159E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X159E-X188D-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X158E-X232V-X238R-X245R-X248D, X022A-X101G-X103A-X104I-X129E-X159E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X148I-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X158E-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X158E-X159E-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X158E-X159E-X232V-X238R-X245R-X248D, X022A-X101G-X103A-X104I-X129E-X158E-X159E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129E-X158E-X188D-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X159E-X232V-X245R-X248D, X101G-X103A-X104I-X188D-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X158E-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X148I-X158E-X232V-X245R-X248D, X101G-X103A-X104I-X129E-X158E-X159E-X232V-X238R-X245R-X248D, X022A-X101G-X103A-X104I-X159E-X188D-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X159E-X188D-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X129E-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129E-X158E-X159E-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X158E-X159E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129E-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X159E-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X128N-X129E-X232V-X245R-X248D, X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129E-X159E-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X158E-X159E-X188D-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X129E-X148I-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X158E-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X128N-X129E-X232V-X238R-X245R-X248D, X022A-X101G-X103A-X104I-X128N-X159E-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X128N-X129E-X158E-X232V-X238R-X245R-X248D, X101G-X103A-X104I-X128N-X129E-X188D-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X128N-X129E-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128N-X158E-X159E-X188D-X232V-X245R-X248D, X022A-X024K-X101G-X103A-X104I-X128N-X158E-X159E-X232V-X245R-X248D, X101G-X103A-X104I-X129E-X148I-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X148I-X232V-X245R-X248D, X022A-X101G-X103A-X104I-X148I-X188D-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X128N-X129E-X188D-X232V-X245R-X248D, X101G-X103A-X104I-X128N-X129E-X158E-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X148I-X158E-X188D-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X148I-X158E-X188D-X232V-X245R-X248D, X101G-X103A-X104I-X148I-X159E-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D, and X101G-X103A-X104I-X128N-X129E-X158E-X232V-X245R-X248D-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
16 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X022A-X024R-X101G-X103A-X104I-X158E-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X129E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X158E-X159E-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X158E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X159E-X188D-X232V-X245R-X248D-X249R, X022A-X024R-X101G-X103A-X104I-X158E-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X159E-X188D-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X148I-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X148I-X158E-X232V-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D-X249R, X016S-X024R-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129E-X159E-X232V-X238R-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X188D-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X129E-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X148I-X158E-X232V-X245R-X248D, X024R-X101G-X103A-X104I-X129E-X188D-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X158E-X159E-X188D-X232V-X238R-X245R-X248D, X022A-X101G-X103A-X104I-X158E-X159E-X232V-X238R-X245R-X248D, X022A-X024R-X101G-X103A-X104I-X129E-X158E-X232V-X245R-X248D, X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X022A-X101G-X103A-X104I-X158E-X159E-X232V-X245R-X248D-X249R, and X024R-X101G-X103A-X104I-X158E-X159E-X232V-X245R-X248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
17 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X104L-X128N-X158E-X186H-X249R, X128N-X158E-X188D-X249R, X062E-X128N-X158E-X159E-X271F, X062E-X158E-X188D-X249R-X271F, X062E-X158E-X186H-X249R-X271F, X128N-X158E-X188D-X209E-X271F, X062E-X159E-X188D-X249R, X016S-X062E-X158E-X186H-X249R, X062E-X158E-X159E-X249R, X101A-X128N-X158E-X209E-X249R, X128N-X158E-X186H-X271F, X062E-X158E-X188D-X249R, X062E-X158E-X186H-X271F, X062E-X158E-X186H-X249R, X062E-X101A-X186H-X249R, X062E-X101A-X158E-X186H-X271F, X062E-X104L-X158E-X188D-X249R-X271F, X062E-X159E-X186H-X249R, X062E-X159E-X249R, X128N-X158E-X186H-X249R, X128N-X158E-X188D-X271F, X062E-X158E-X249R, X062E-X186H-X188D-X249R-X271F, X128N-X158E-X209E-, X062E-X101A-X158E-X249R, X104L-X128N-X158E-X186H-X271F, X062E-X101A-X158E-X186H-X249R-X271F, X016S-X062E-X158E-X249R, X062E-X101A-X159E-X249R, X128N-X158E-X186H-X188D-X271F, X101A-X128N-X158E-X186H-X271F, X062E-X101A-X188D-X249R, X101A-X104L-X158E-X186H-X188D-X249R, X062E-X159E-X249R-X271F, X128N-X158E-X159E-X271F, X016S-X062E-X104L-X158E-X186H-X271F, X022A-X128N-X158E-X249R, X128N-X158E-X249R, X062E-X101A-X104L-X158E-X186H-X271F, X016S-X062E-X158E-X186H-X271F, X104L-X128N-X158E-X249R, X104L-X128N-X158E-X188D-X249R, X022A-X062E-X158E, X062E-X101A-X188D-X249R-X271F, X062E-X158E-X249R-X271F, X104L-X128N-X158E-X186H-X188D-X271F, X062E-X101A-X186H-X271F, X062E-X104L-X159E-X249R, X062E-X186H-X249R, X062E-X101A-X186H-X249R-X271F, X101A-X158E-X186H-X188D-X249R, X062E-X101A-X186H, X101A-X128N-X129E-X186H-X249R, X101A-X103G-X158E-X186H-X249R, X016S-X062E-X104L-X186H-X188D-X271F, X104L-X158E-X186H-X249R, X101A-X128N-X158E-X188D-X209E-X271F, X062E-X101A-X186H-X188D-X271F, X016S-X062E-X158E-X249R-X271F, X062E-X128N-X158E, X062E-X128N-X159E-X249R, X062E-X101A-X158E-X188D-X249R, X101A-X128N-X158E-X249R, X062E-X158E-X186H-X188D-X249R, X016S-X104L-X158E-X186H-X271F, X062E-X148I-X159E, X062E-X101A-X158E-X186H-X249R, X062E-X101A-X186H-X188D-X249R, X104L-X158E-X186H-X188D-X249R, X062E-X101A-X104L-X186H-X188D-X271F, X022A-X101A-X158E-X186H-X249R, X101A-X128N-X158E-X209E, X158E-X186H-X188D-X249R-X271F, X104L-X158E-X186H-X188D-X249R-X271F, X101A-X104L-X158E-X186H-X249R, X104L-X158E-X249R, X101A-X104L-X128N-X158E-X186H-X271F, X016S-X104L-X188D-X249R, X101A-X104L-X158E-X186H-X188D-X271F, X104L-X128N-X159E-X271F, X104L-X158E-X186H-X249R-X271F, X158E-X186H-X249R, X101A-X158E-X186H-X249R, X104L-X158E-X188D-X249R-X271F, X016S-X128N-X158E-X186H, X104L-X128N-X186H-X188D-X249R, X016S-X101A-X128N-X186H, X016S-X062E-X128N-X186H-X271F, X016S-X128N-X186H-X271F, X128N-X129E-X186H, X158E-X186H-X249R-X271F, X016S-X158E-X249R, X016S-X158E-X186H-X249R, X016S-X022A-X158E-X186H-X271F, X089P-X101A-X129E-X186H, X022A-X128N-X158E-X186H, X101A-X104L-X128N-X158E-X186H, X022A-X128N-X186H-X188D-, X062E-X104L-X158E-X186H-X188D-X249R, X022A-X158E-X186H-X249R-X271F, X022A-X104L-X158E-X249R, X101A-X111V-X129E, X016S-X158E-X016S-X111V-X188D-, X022A-X104L-X186H-X188D-X249R, and X104L-X148I-X188D-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
18 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X001R-X101G-X103A-X104I-X232V-X245R, X004R-X101G-X103A-X104I-X232V-X245R, X043R-X101G-X103A-X104I-X232V-X245R-X271L, X078R-X101G-X103A-X104I-X232V-X245R, X004R-X043R-X101G-X103A-X104I-X232V-X245R, X018R-X043R-X101G-X103A-X104I-X232V-X245R, X020R-X101G-X103A-X104I-X232V-X245R, X101G-X103A-X104I-X232V-X245R-X271L, X020R-X043R-X101G-X103A-X104I-X232V-X245R, X024R-X043R-X101G-X103A-X104I-X232V-X245R, X020R-X025R-X116A-X167W, X018R-X101G-X103A-X104I-X232V-X245R, X022R-X101G-X103A-X104I-X232V-X245R, X078R-X103N-X106G-X167W-X236N, X018R-X043D-X101G-X103A-X104I-X232V-X245R-X269R, X043R-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X101A-X120E-X194F-X249R, X020R-X043D-X101G-X103A-X104I-X232V-X245R-X269R, X101G-X103A-X104I-X212F-X232V-X245R, X020R-X144R-X185I-X233C-X236N, X023A-X078R-X216F-X236N-X249R, X101G-X103A-X104I-X232V-X245R-X269R, X101G-X103A-X104I-X115R-X232V-X245R, X052N-X078R-X103N-X148I-X213A, X018R-X043D-X101G-X103A-X104I-X232V-X245R-X249R, X024R-X043D-X101G-X103A-X104I-X232V-X245R-X249R, X024R-X043D-X101G-X103A-X104I-X232V-X245R-X269R, X025R-X089I-X116A-X239S-X270C, X024R-X148I-X213A-X252R, X024R-X025R-X183D-X192W-X239S, X046R-X194F-X212M, X104L-X217E-X224A-X249R-X252R, X023A-X091F-X121F-X192W-X236N, X101G-X103A-X104I-X232V-X244R-X245R, X099F-X144R-X167W-X252R, X101G-X103A-X104I-X232V-X245R-X249R, X043R-X101G-X103A-X104I-X232V-X245R, X022W-X078R-X167W-X212M-X270C, X121F-X252R-X270C, X020R-X103N-X216F-X236N-X252R, X043R-X101G-X103A-X104I-X232V-X245R-X249R, X023A-X052N-X192W-X198L-X252R, X025R-X046R-X121F, X024R-X078R-X104L-X116A-X183D, X046R-X059A-X103N-X211Q-X212M, X020R-X052N-X062Q-X091F-X192W, X023A-X052N-X144R-X192W-X216F, X101G-X103A-X104I-X232V-X242R-X245R, X052N-X103N-X116A-X148I-X192W, X089I-X116A-X117F-X224A-X249R, X144R-X211Q-X238L-X239S-X249R, X043A-X062Q-X194F-X211Q, X020R-X024R-X052N-X059A-X216F, X024R-X167W-X224A-X249R, X057R-X167W-X249R, X025R-X103N-X186K-X194F-X224A, X105T-X128N-X144R-X148I-X212M, X020R-X059A-X144R-X192W-X224A, X024R-X043A-X117F-X194F-X211Q, X117F-X194F-X213A-X270C, X078R-X091F-X121F-X233C-X252R, X057R-X099F-X105T-X198L-X213A, X023A-X091F-X101A-X198L-X252R, X062Q-X103N-X121F-X144R-X249R, X043R-X101G-X103A-X104I-X232V-X242R-X245R, X023A-X024R-X117F-X212M-X216F, X104L-X213A-X216F, X194F-X211Q-X236N, X062Q-X103N-X117F-X194F, X024R-X062Q-X104L-X106G-X249R, X057R-X089I-X198L, X046R-X059A-X106G-X217E-X249R, X117F-X213A-X215F, X101A-X120E-X192W-X215F-X224A, X043A-X057R-X117F-X144R-X183D, X046R-X183D-X238L, X025R-X043A-X089I-X117F, X078R-X104L-X213A-X215F-X224A, X091F-X099F-X101A-X105T-X167W, X106G-X117F-X238L, X046R-X089I-X091F-X101A-X116A, X020R-X062Q-X089I-X186K-X212M, X057R-X099F-X121F-X185I-X192W, X046R-X089I-X192W-X233C-X270C, X089I-X117F-X185I-X215F-X233C, X052N-X104L-X183D-X216F-X249R, X078R-X099F-X116A-X186K-X224A, X025R-X105T-X128N-X144R-X270C, X105T-X211Q-X216F, X024R-X046R-X091F-X121F, X106G-X185I-X216F-X236N, X062Q-X101A-X236N-X252R-X270C, X025R-X043A-X091F-X198L-X270C, X020R-X023A-X104L-X192W-X233C, X024R-X043A-X105T-X106G-X198L, X020R-X089I-X217E, X024R-X091F-X198L-X215F-X239S, X046R-X089I-X099F-X186K-X212M, X104L-X120E-X186K-X216F-X252R, X022W-X194F-X213A-X233C-X238L, X099F-X105T-X106G-X194F-X212M, X089I-X105T-X116A-X215F-X216F, X025R-X116A-X120E-X224A-X270C, X043A-X059A-X101A-X216F-X224A, X057R-X183D-X236N, X025R-X062Q-X128N-X144R-X185I, X103N-X120E-X167W-X198L-X233C, X022W-X089I-X216F, X024R-X106G-X116A-X212M-X224A, X020R-X052N-X101A-X198L-X233C, X089I-X091F-X185I-X211Q-X270C, X111I-X215F-X239S, X024R-X116A-X186K-X233C-X236N, and X023A-X103N-X106G-X212M-X215F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
19 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020R-X022W-X078R-X101A-X103A-X104I-X116S-X213A-X215F-X232V-X245R, X018R-X078R-X101G-X103A-X104I-X232V-X245R, X024R-X045T-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X022W-X078R-X101G-X103A-X104I-X116A-X232V-X245R, X020R-X22W-X101G-X103A-X104I-X232V-X245R, X018R-X043R-X101G-X103A-X104I-X232V-X245R, X018R-X022W-X024R-X076D-X101A-X116A-X232V-X245R, X018R-X104I-X232V-X249R, X018R-X024R-X076D-X101A-X116A-X211Q-X249R, X018R-X043D-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X018R-X043R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X045T-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X043D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X043D-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X103A-X232V-X249R, X018R-X101G-X104I-X232V-X245R, X020R-X024R-X101G-X103A-X104I-X217E-X232V-X245R-X249R, X018R-X22K-X043D-X101G-X103A-X104I-X232V-X245R, X043R-X045T-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X22W-X101G-X103A-X104I-X211Q-X232V-X245R, X024R-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X22W-X078R-X101A-X103A-X104I-X116A-X183D-X232V, X018R-X024R-X076D-X116A-X215F-X249R, X018R-X043R-X045T-X101G-X103A-X104I-X232V-X245R, X024R-X043R-X076D-X101G-X103A-X104I-X232V-X245R, X020R-X022W-X101G-X103A-X104I-X232V-X245R, X020R-X022W-X101G-X103A-X104I-X211Q-X232V-X245R, X020R-X022W-X078R-X101G-X103A-X104I-X116A-X213A-X215F-X232V-X245R, X043D-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X024R-X076D-X101A-X116A-X213A-X249R, X018R-X024R-X076D-X116A-X211Q-X249R, X043R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X101G-X245R, X020R-X22W-X101A-X103A-X104I-X211Q-X213A-X232V-X245R, X020R-X024R-X043D-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X020R-X078R-X101G-X103A-X104I-X211Q-X213A-X215F-X232V-X245R, X045T-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X043D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X101G-X103A-X249R, X018R-X22W-X024R-X076D-X101A-X116A-X232V-X245R, X018R-X101G-X104I-X232V-X249R, X020R-X22W-X101A-X103A-X104I-X215F-X232V-X245R, X018R-X024R-X076D-X211Q-X213A-X249R, X018R-X022W-X024R-X076D-X101A-X198L-X249R, X024R-X101G-X103A-X104I-X232V-X245R, X020R-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X101G-X103A-X104I-X232V-X245R-X269R, X043D-X078R-X101G-X103A-X104I-X232V-X245R, X020R-X101G-X104I-X213A-X215F-X232V-X245R, X020R-X101G-X103A-X104I-X116A-X215F-X232V-X245R, X024R-X103A-X104I-X249R, X018R-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X101G-X104I-X245R, X020R-X101G-X103A-X104I-X211Q-X213A-X215F-X232V-X245R, X024R-X103A-X104I-X232V-X249R, X018R-X024R-X076D-X116A-X211Q-X215F-X249R, X018R-X245R, X024R-X103A-X245R, X024R-X103A-X104I-X245R, X020R-X078R-X101G-X232V-X245R, X018R-X024R-X076D-X104I-X249R, X018R-X024R-X104I-X249R, X024R-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X076D-X211Q-X215F-X249R, X019H-X020R-X022W-X078R-X101G-X103A-X104I-X211Q-X232V-X245R, X018R-X024R-X076D-X101A-X198L-X211Q-X213A-X249R, X018R-X024R-X043D-X101G-X103A-X104I-X232V-X245R, X020R-X22W-X103A-X104I-X232V-X245R, X018R-X103A-X104I-X249R, X018R-X022W-X024R-X076D-X101A-X198L-X215F-X249R, X018R-X024R-X101G-X104I-X232V, X078R-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X043R-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X020R-X043D-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X22W-X024R-X076D-X116A-X213A-X249R, X018R-X024R-X101G-X104I, X020R-X101A-X103A-X104I-X215F-X232V-X245R, X018R-X045T-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X101G-X103A-X245R, X043R-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X211Q-X215F-X232V-X245R, X020R-X22W-X078R-X101G-X103A-X104I-X116A-X213A-X215F-X232V-X245R, X020R-X078R-X101G-X103A-X104I-X215F-X232V-X245R, X020R-X022W-X078R-X101G-X103A-X104I-X116A-X183D-X232V-X245R, X076D-X101G-X103A-X104I-X232V-X245R, X076D-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X22W-X101A-X103A-X104I-X232V-X245R, X020R-X101G-X103A-X232V-X245R, X020R-X022W-X078R-X101A-X103A-X104I-X116A-X183D-X232V-X245R, X018R-X020R-X024R-X045T-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X043R-X045T-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X101G-X104I-X249R, X020R-X22W-X078R-X101G-X103A-X104I-X116A-X183D-X232V-X245R, X020R-X022W-X101G-X103A-X104I-X198L-X211Q-X213A-X232V-X245R, X020R-X078R-X101A-X103A-X104I-X116A-X183D-X213A-X232V-X245R, X024R-X076D-X104I-X232V-X245R, X018R-X020R-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X076D-X101G-X104I-X232V-X249R, X018R-X043D-X078R-X101G-X103A-X104I-X232V-X245R, X001T-X018R-X024R-X076D-X116A-X213A-X249R, X076D-X078R-X101G-X103A-X104I-X232V-X245R, X020R-X078R-X101G-X103A-X104I-X116A-X232V-X245R, X043R-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X045T-X101G-X103A-X104I-X232V-X245R, X018R-X076D-X101G-X104I-X232V-X245R, X020R-X078R-X101G-X103A-X104I-X116A-X183D-X232V-X245R, X018R-X024R-X076D-X101A-X211Q-X213A-X215F-X249R, X045T-X078R-X101G-X103A-X104I-X232V-X245R, X043R-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X022W-X078R-X101G-X103A-X104I-X116A-X183D-X213A-X232V-X245R, X020R-X022W-X101G-X103A-X104I-X116A-X183D-X213A-X232V-X245R, X020R-X101G-X198L-X215F-X232V-X245R, X018R-X024R-X076D-X213A-X215F-X249R, X020R-X078R-X101G-X103A-X104I-X116A-X211Q-X232V-X245R, X020R-X022W-X078R-X101A-X103A-X104I-X116A-X183D-X215F-X232V-X245R, X020R-X022W-X078R-X101A-X103A-X104I-X116A-X183D-X213A-X232V-X245R, X024R-X232V-X245R, X018R-X024R-X043D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X024R-X076D-X101A-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X213A-X249R, X101G-X103A-X104I-X232V-X245R, X018R-X024R-X076D-X211Q-X213A-X215F-X249R, X018R-X024R-X076D-X116A-X213A-X215F-X249R, X043D-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X043D-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X020R-X022W-X078R-X101G-X103A-X104I-X116A-X183D-X211Q-X232V-X245R, X018R-X024R-X076D-X101A-X198L-X211Q-X215V-X249R, X018R-X022W-X024R-X076D-X116A-X211Q-X249R, X020R-X103A-X104I-X232V-X245R, X020R-X22W-X101A-X103A-X104I-X211Q-X215F-X232V-X245R, X024R-X076D-X101G-X103A-X104I-X232V-X245R, X020R-X101G-X103A-X104I-X116A-X232V-X245R, X018R-X22W-X024R-X076D-X198L-X215F-X249R, X020R-X022W-X103A-X104I-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X211Q-X213A-X232V-X245R, X020R-X022W-X101G-X103A-X104I-X211Q-X213A-X215F-X232V-X245R, X018R-X043R-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X022W-X024R-X076D-X101A-X198L-X213A-X215F-X249R, X018R-X22W-X024R-X076D-X101A-X215F-X249R, X020R-X045T-X101G-X103A-X104I-X232V-X245R, X020R-X078R-X101G-X103A-X104I-X180A-X232V-X245R, X020R-X022W-X078R-X101G-X103A-X104I-X116A-X183D-X215F-X232V-X245R, X018R-X101G-X103A-X104I, X020R-X22W-X101A-X103A-X104I-X116A-X211Q-X213A-X215F-X232V-X245R, X018R-X024R-X076D-X103A-X232V-X245R, X020R-X078R-X101A-X103A-X104I-X116A-X183D-X232V-X245R, X018R-X024R-X076D-X211Q-X249R, X020R-X101A-X103A-X104I-X213A-X215F-X232V-X245R, X018R-X020R-X043D-X101G-X103A-X104I-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X211Q-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X116A-X211Q-X215F-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X116A-X211Q-X232V-X245R, X018R-X024R-X076D-X232V-X249R, X018R-X024R-X045T-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X076D-X245R, X024R-X104I-X245R, X101G-X232V, X020R-X22W-X101A-X103A-X104I-X116A-X211Q-X215F-X232V-X245R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X198L-X213A-X249R, X020R-X022W-X078R-X101A-X103A-X104I-X232V-X245R, X020R-X022W-X078R-X101A-X103A-X104I-X198L-X232V-X245R, X024R-X245R-X249R, X020R-X101G-X103A-X104I-X116A-X211Q-X232V-X245R, X018R-X22W-X024R-X076D-X116A-X211Q-X249R, X024R-X101G-X104I-X249R, X018R-X024R-X076D-X116A-X198L-X211Q-X213A-X215F-X249R, X018R-X024R-X104I, X020R-X022W-X078R-X101A-X103A-X104I-X183D-X198L-X213A-X215F-X232V-X245R, X018R-X020R-X22W-X024R-X076D-X116A-X183D-X198L-X213A-X249R, X020R-X022W-X101A-X103A-X104I-X232V-X245R, X012H-X020R-X078R-X101G-X103A-X104I-X198L-X211Q-X213A-X232V-X245R, X018R-X024R-X076D-X101A-X211Q-X215F-X249R, X018R-X024R-X076D-X213A-X249R, X024R-X104I-X249R, X018R-X022W-X024R-X076D-X211Q-X249R, X018R-X076D-X103A-X104I-X249R, X043R-X076D-X101G-X103A-X104I-X217E-X232V-X245R, X020R-X078R-X101G-X103A-X104I-X183D-X211Q-X213A-X232V-X245R-X271G, X018R-X022W-X024R-X076D-X101A-X116A-X198L-X215F-X249R, X018R-X024R-X043D-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X076D-X101A-X198L-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X198L-X211Q-X213A-X249R, X018R-X024R-X076D-X101A-X116A-X213A-X215F-X249R, X020R-X043D-X045T-X078R-X101G-X103A-X104I-X232V-X245R, X045T-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X22W-X024R-X076D-X101A-X116A-X215F-X249R, X043D-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X022W-X078R-X101A-X103A-X104I-X116A-X183D-X213A-X215F-X232V-X245R, X018R-X024R-X076D-X116A-X211Q-X213A-X215F-X249R, X024R-X103A-X175L-X232V-X249R, X018R-X024R-X076D-X116A-X213A-X249R, X020R-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X020R-X078R-X101A-X103A-X104I-X116A-X183D-X211Q-X213A-X215F-X232V-X245R, X024R-X043R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X076D-X101A-X116A-X198L-X213A-X215F-X249R, X043R-X045T-X101G-X103A-X104I-X232V-X245R, X018R-X022W-X024R-X076D-X101A-X198L-X211Q-X245R, X020R-X024R-X043D-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X232V, X020R-X078R-X101A-X103A-X104I-X116A-X183D-X211Q-X232V-X245R, X024R-X076D-X101G-X232V-X245R, X018R-X076D-X101G-X103A-X104I-X249R, X018R-X024R-X076D-X198M-X211Q-X213A-X215F-X249R, X018R-X22W-X024R-X076D-X101A-X198L-X215F-X249R, X018R-X043R-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X020R-X022W-X103A-X211Q-X213A-X215F-X232V-X245R, X020R-X078R-X101A-X103A-X104I-X116A-X183D-X211Q-X213A-X232V-X245R, X045T-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X22W-X024R-X076D-X101A-X211Q-X213A-X249R, X018R-X024R-X076D-X116A-X198L-X215F-X249R-X268G, X018R-X022W-X024R-X076D-X101A-X211Q-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X198L-X211Q-X249R, X101G-X103A-X104I-X245R, X018R-X076D-X101G-X232V-X245R, X018R-X020R-X045T-X101G-X103A-X104I-X232V-X245R, X018R-X022W-X024R-X076D-X101A-X116A-X198L-X249R, X018R-X022W-X024R-X076D-X116A-X198L-X213A-X249R, X078R-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X020R-X043R-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X043D-X076D-X101G-X103A-X104I-X232V-X245R-X249R, X045T-X101G-X103A-X104I-X232V-X245R-X269R, X024R-X103A-X232V, X018R-X024R-X076D-X101A-X211Q-X249R, X043D-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X043R-X045T-X076D-X076T-X101G-X103A-X104I-X232V-X245R-X273T, X018R-X020R-X043D-X078R-X101G-X103A-X104I-X232V-X245R, X020R-X078R-X101A-X103A-X104I-X183D-X215F-X232V-X245R, X018R-X024R-X076D-X101G-X232V, X232V-X245R, X043R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X234I-X245R, X024R-X076D-X103A-X104I-X245R, X020R-X078R-X101A-X103A-X104I-X211Q-X213A-X232V-X245R, X043D-X078R-X101G-X103A-X104I-X232V-X245R-X269R-X272V, X018R-X020R-X024R-X076D-X116A-X183D-X198L-X211Q-X249R, X018R-X103A-X104I-X232V, X020R-X022W-X101G-X103A-X104I-X116A-X183D-X213A-X215F-X232V-X245R, X024R-X043R-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X022W-X024R-X076D-X213A-X215F-X249R, X018R-X024R-X076D-X101A-X116A-X198L-X211Q-X213A-X249R, X020R-X22W-X078R-X101A-X103A-X104I-X116A-X183D-X213A-X232V-X245R, X020R-X022W-X078R-X101G-X103A-X104I-X211Q-X213A-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X215F-X232V-X245R, X018R-X020R-X024R-X076D-X116A-X183D-X215F-X249R, X018R-X024R-X076D-X116A-X117I-X183D-X213A-X249R, 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X018R-X024R-X076D-X101A-X183D-X198L-X211Q-X213A-X249R, X018R-X043R-X045T-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X018R-X024R-X076D-X183D-X211Q-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X215F-X249R, X018R-X020R-X024R-X076D-X116A-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X211Q-X215F-X249R, X018R-X020R-X024R-X076D-X183D-X211Q-X213A-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X198L-X213A-X249R, X018R-X020R-X024R-X076D-X101A-X211Q-X215F-X249R-X269D, X018R-X020R-X043D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X022W-X024R-X076D-X101A-X183D-X198L-X213A-X249R, X020R-X022W-X101G-X103A-X104I-X183D-X215F-X232V-X245R, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X209H-X211Q-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X183D-X211Q-X249R, X076D-X232V-X245R, X043D-X045T-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X020R-X024R-X076D-X116A-X198L-X211Q-X215F-X245R, X018R-X020R-X022W-X024R-X076D-X213A-X249R, X020R-X024R-X043D-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X198L-X215F-X249R, X020R-X090S-X101G-X103A-X104I-X116A-X183D-X213A-X215F-X232V-X245R, X018R-X020R-X022W-X024R-X076D-X101A-X198L-X211Q-X213A-X215F-X249R, X018R-X020R-X024R-X076D-X101A-X183D-X213A-X215F-X249R, X018R-X024R-X076D-X116A-X183D-X198L-X211Q-X213A-X249R, X043D-X045T-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X024R-X076D-X101A-X183D-X215F-X249R, X018R-X020R-X024R-X076D-X101A-X183D-X198L-X213A-X249R, X020R-X022W-X101A-X103A-X104I-X116A-X183D-X198L-X211Q-X213A-X215F-X232V-X245R, X018R-X020R-X024R-X043D-X076D-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X024R-X076D-X101A-X183D-X213A-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X198L-X211Q-X213A-X215F-X249R, X018R-X020R-X043D-X101G-X103A-X104I-X217E-X232V-X245R, X076D-X103A-X104I-X232V-X245R, X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X022W-X024R-X076D-X101A-X183D-X211Q-X215F-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X249R, X018R-X024R-X076D-X116A-X183D-X198L-X211Q-X215F-X249R, X018R-X020R-X043D-X076D-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X076D-X103A-X104I-X245R, X018R-X020R-X024R-X076D-X101A-X116A-X211Q-X213A-X215F-X249R, X018R-X024R-X076D-X101A-X183D-X198L-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X211Q-X215F-X249R, X018R-X024R-X076D-X116A-X183D-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X211Q-X249R, X018R-X024R-X076D-X101G-X245R, X018R-X024R-X076D-X101A-X116A-X183D-X198L-X213A-X215F-X249R, X043R-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X020R-X022W-X024R-X076D-X116A-X198L-X213A-X215F-X249R, X076D-X104I-X232V-X245R, X027R-X043R-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X022W-X024R-X076D-X101A-X183D-X213A-X215F-X249R, X018R-X024R-X076D-X103A-X104I-X135I-X232V, X018R-X020R-X022W-X024R-X076D-X183D-X198L-X211Q-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X215F-X249R, X018R-X024R-X076D-X101A-X116A-X183D-X211Q-X249R, X018R-X022W-X024R-X076D-X183D-X211Q-X213A-X215F-X249R, X076D-X101G-X103A-X104I-X232V-X249R, X005S-X018R-X022W-X024R-X076D-X101A-X213A-X215F-X249R, X018R-X024R-X076D-X116A-X183D-X215F-X249R, X018R-X024R-X076D-X183D-X198L-X215F-X249R, X018R-X020R-X024R-X076D-X101A-X116A-X183D-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X211Q-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X213A-X249R, X018R-X022W-X024R-X076D-X101A-X183D-X211Q-X213A-X249R, X018R-X020R-X024R-X076D-X116A-X198L-X249R, X018R-X024R-X076D-X183D-X211Q-X213A-X249R, X018R-X024R-X076D-X101A-X116A-X183D-X213A-X249R, X018R-X024R-X076D-X116A-X183D-X198L-X215F-X249R, X018R-X022W-X024R-X076D-X183D-X198L-X211Q-X215F-X249R, X018R-X022W-X024R-X076D-X183D-X249R, X018R-X022W-X024R-X076D-X101A-X183D-X198L-X215F-X249R, X018R-X020R-X043D-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X020R-X022W-X101A-X103A-X104I-X183D-X198L-X215F-X232V-X245R, X076D-X101G-X103A-X104I-X249R, X018R-X020R-X024R-X076D-X183D-X198L-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X183D-X215F-X249R, X018R-X020R-X024R-X076D-X211Q-X213A-X249R, X018R-X024R-X076D-X116A-X183D-X211Q-X213A-X249R, X018R-X024R-X076D-X116A-X183D-X198L-X249R, X018R-X020R-X024R-X076D-X211Q-X243D-X249R, X018R-X020R-X045T-X076D-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X022W-X024R-X076D-X183D-X198L-X211Q-X213A-X249R, X018R-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X213A-X249R, X018R-X022W-X024R-X076D-X116A-X198L-X211Q-X213A-X215F-X249R, X024R-X076D-X101G, X018R-X020R-X024R-X076D-X101A-X198L-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X211Q-X213A-X249R, X018R-X020R-X024R-X076D-X183D-X215F-X249R, X018R-X043D-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X211Q-X213A-X245R, X020R-X024R-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X020R-X045T-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X024R-X076D-X101A-X183D-X198L-X211Q-X249R, X018R-X022W-X024R-X076D-X183D-X211Q-X213A-X249R, X018R-X020R-X024R-X076D-X116A-X198L-X211Q-X215F-X249R-X269S, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X215F-X249R, X020R-X022W-X101A-X103A-X104I-X183D-X213A-X215F-X232V-X245R, X018R-X024R-X076D-X086V-X101A-X183D-X198L-X211Q-X249R, X018R-X076D-X101G-X198T-X232V, X018R-X020R-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X215F-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X101A-X183D-X211Q-X213A-X215F-X249R, X018R-X024R-X076D-X183D-X249R-X248T, X018R-X076D-X104I-X245R-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X213A-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X211Q-X213A-X215F-X249R, X018R-X020R-X024R-X076D-X101A-X249R, X018R-X020R-X024R-X076D-X101A-X116A-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X211Q-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X211Q-X213A-X249R, X043R-X045T-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X043R-X045T-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X076D-X101G-X104I, X020R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X249R, X043D-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X249R, X018R-X076D-X104I, X018R-X020R-X043D-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X020R-X022W-X024R-X076D-X183D-X211Q-X249R, X018R-X024R-X076D-X101A-X183D-X198L-X211Q-X215F-X249R, X101G-X104I, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X211Q-X213A-X215F-X249R, X018R-X020R-X024R-X076D-X101A-X183D-X211Q-X213A-X215F-X249R, X018R-X020R-X024R-X076D-X116A-X183D-X215F-X249R-X263D, X018R-X022W-X024R-X076D-X101A-X183D-X249R, X018R-X024R-X076D-X101A-X116A-X183D-X198L-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X198L-X215F-X249R-X269H, X018R-X043D-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X045T-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X078R-X101G-X103A-X104I-X217E-X232V-X245R, X018R-X020R-X022W-X024R-X076D-X101A-X183D-X198L-X213A-X215F-X249R, X018R-X020R-X024R-X076D-X183D-X198L-X213A-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X198L-X211Q-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X211Q-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X198L-X211Q-X213A-X249R, X018R-X020R-X022W-X024R-X076D-X249R, X020R-X022W-X101G-X103A-X104I-X116A-X183D-X198L-X211Q-X213A-X215F-X232V-X245R, X104I-X232V, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X183D-X211Q-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X213A-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X183D-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X101A-X183D-X198L-X211Q-X213A-X249R, X018R-X022W-X024R-X076D-X101A-X183D-X198L-X249R, X043D-X045T-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X020R-X024R-X076D-X101A-X183D-X198L-X211Q-X249R, X018R-X020R-X043R-X101G-X103A-X104I-X217E-X232V-X245R, X024R-X043R-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X043R-X045T-X076D-X078R-X101G-X103A-X104I-X166F-X176P-X179V-X184T-X187P-X194P, X018R-X022W-X024R-X041E-X076D-X101A-X160T-X183D-X211Q-X213A-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X198L-X211Q-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X198L-X213A-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X213A-X249R, X018R-X020R-X024R-X076D-X198L-X213A-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X198L-X211Q-X249R, X018R-X020R-X024R-X076D-X183D-X211Q-X215F-X249R, X024R-X076D-X104I, X018R-X076D-X101G-X232V, X018R-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X213A-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X215F-X249R, X018R-X024R-X031F-X076D-X116A-X183D-X211Q-X213A-X215F-X249R, X018R-X024R-X076D-X101A-X116A-X183D-X198L-X213A-X249R-X269S, X018R-X020R-X024R-X076D-X101A-X116A-X198L-X215F-X249R, X018R-X043R-X076D-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X020R-X024R-X076D-X101A-X114T-X198L-X211Q-X215F-X249R, X024R-X043R-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X024R-X076D-X103A-X104I-X232V, X018R-X020R-X022W-X024R-X076D-X101A-X198L-X211Q-X215F-X249R, X101G-X104I-X232V, X018R-X022W-X024R-X076D-X183D-X198L-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X183D-X198L-X211Q-X249R, X018R-X020R-X024R-X076D-X183D-X213A-X249R, X018R-X024R-X076D-X101A-X183D-X211Q-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X211Q-X213A-X249R, X018R-X078R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X211Q-X215F-X249R-X260A, X076D-X249R, X018R-X022W-X024R-X076D-X183D-X211Q-X215F-X249R, X018R-X020R-X024R-X076D-X116A-X211Q-X215F-X249R, X018R-X020R-X024R-X076D-X213A-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X183D-X198L-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X183D-X249R, X076D-X104I-X249R, X020R-X022W-X101A-X103A-X104I-X116A-X183D-X211Q-X213A-X215F-X232V-X245R, X024R-X043R-X045T-X078R-X101G-X103A-X104I-X217E-X232V-X245R, X018R-X020R-X022W-X024R-X076D-X183D-X198L-X215F-X249R, X018R-X076D-X101G-X103A, X020R-X024R-X101G-X103A-X104I-X232V-X245R-X269R, X018R-X020R-X024R-X076D-X101A-X116A-X183D-X211Q-X249R, X101G-X103A-X232V, X024R-X076D-X101G-X232V, X018R-X022W-X024R-X076D-X101A-X183D-X198L-X211Q-X249R, X018R-X020R-X024R-X076D-X101A-X183D-X198L-X211Q-X215F-X249R, X018R-X020R-X024R-X076D-X183D-X198L-X249R, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X249R, X018R-X020R-X024R-X076D-X116A-X156V-X183D-X211Q-X215F-X249R-X269S, X045T-X076D-X101G-X103A-X104I-X232V-X245R-X249R, X018R-X020R-X024R-X076D-X101A-X183D-X198L-X249R, X018R-X020R-X022W-X024R-X076D-X198L-X211Q-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X198L-X211Q-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X116A-X249R, X018R-X076D-X101G, X018R-X024R-X076D-X116A-X183D-X198L-X213A-X215F-X249R, X018R-X020R-X022W-X024R-X076D-X101A-X116A-X198L-X213A-X215F-X249R, X018R-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X249R, X018R-X076D-X232V, X018R-X020R-X024R-X076D-X101A-X232V-X245R, X018R-X043D-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X018R-X022W-X024R-X076D-X183D-X213A-X215F-X249R, X018R-X024R-X076D-X101A-X116A-X211Q-X213A-X237D-X249R, X018R-X020R-X024R-X076D-X101A-X116A-X183D-X211Q-X249R-X275S, X018R-X022W-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X213A-X215F-X249R, X024R-X076D, X018R-X024R-X076D-X183D-X211Q-X215F-X249R, X018R-X022W-X024R-X076D-X116A-X183D-X198L-X213A-X215F-X249R, X076D-X104I-X232V-X249R, X018R-X076D-X103A-X232V, X018R-X020R-X024R-X076D-X101A-X116A-X183D-X198L-X211Q-X213A-X249R, X018R-X020R-X024R-X076D-X101A-X183D-X213A-X249R, X018R-X020R-X024R-X076D-X101A-X175E-X183D-X211Q-X215F-X249R, X018R-X020R-X043D-X078R-X101G-X103A-X104I-X217E-X232V-X245R-X273E, X020R-X024R-X043D-X045T-X076D-X101G-X103A-X104I-X232V-X245R, X005S-X101G-X103A-X104I-X232V-X245R-X249R, X103A-X104I-X232V, X018R-X020R-X024R-X068A-X076D-X101A-X116A-X213A-X215F-X249R, X018R-X022W-X024R-X076D-X101A-X198L-X215F-X249R-X275S, X018R-X024R-X076D-X183D-X198L-X211Q-X213A-X249R, and X043D-X045T-X101G-X103A-X104I-X232V-X245R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
20 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X018R-X024R-X043R-X076D-X249R-X269R, X018R-X022R-X024R-X043R-X076D-X249R, X018R-X043D-X101G-X103A-X104I-X232V-X245R, X020R-X043D-X101G-X103A-X104I-X232V-X245R, X043D-X101G-X103A-X104I-X232V-X245R-X269R, X043D-X078R-X101G-X103A-X104I-X232V-X245R, X043R-X076D-X101G-X103A-X104I-X232V-X245R, X022R-X043R-X101G-X103A-X104I-X232V-X245R, X043R-X078R-X101G-X103A-X104I-X232V-X245R, X020R-X076D-X101G-X103A-X104I-X232V-X245R, X043R-X076D-X101G-X103A-X104I-X232V-X245R, X022R-X076D-X101G-X103A-X104I-X232V-X245R, X076D-X078R-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X043R-X076D-X249R, X018R-X024R-X076D-X242R-X249R, X018R-X024R-X076D-X249R-X269R, X018R-X022R-X024R-X076D-X249R, X018R-X024R-X076D-X078R-X249R, X018R-X024R-X043D-X076D-X249R-X269R, X018R-X022R-X024R-X043D-X076D-X249R, X018R-X024R-X043D-X076D-X078R-X249R, X020R-X101G-X103G-X104I-X232V-X245R, X020R-X101G-X103A-X104L-X232V-X245R, X020R-X101G-X103A-X104V-X232V-X245R, X020R-X101G-X103S-X104I-X232V-X245R, X020R-X101G-X103S-X104L-X232V-X245R, X020R-X101S-X103S-X104I-X232V-X245R, X020R-X101S-X103S-X104L-X232V-X245R, X020R-X101A-X103A-X104L-X232V-X245R, X020R-X101S-X103S-X104V-X232V-X245R, X020R-X101S-X103A-X104I-X232V-X245R, X020R-X101S-X103A-X104V-X232V-X245R, X020R-X101S-X103G-X104I-X232V-X245R, X020R-X101S-X103G-X104V-X232V-X245R, X020R-X101A-X103A-X104V-X232V-X245R, X020R-X101A-X103S-X104I-X232V-X245R, X020R-X101A-X103S-X104V-X232V-X245R, X018R-X024R-X043R-X076D-X078R-X249R, X024R-X043D-X101G-X103A-X104I-X232V-X245R, X043D-X101G-X103A-X104I-X232V-X245R-X249R, X024R-X076D-X101G-X103A-X104I-X232V-X245R, X076D-X101G-X103A-X104I-X232V-X242R-X245R, X018R-X020R-X024R-X076D-X217E-X249R, X018R-X024R-X043R-X076D-X217E-X249R, X018R-X024R-X043D-X076D-X242R-X249R, X018R-X020R-X024R-X043R-X076D-X249R, X020R-X101A-X103G-X104V-X232V-X245R, X043D-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X076D-X217E-X249R-X269R, and X018R-X024R-X076D-X217E-X242R-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
21 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020R-X101A-X103A-X104I-X118R-X232V-X245R, X020R-X024R-X116A-X213A, X043R-X101A-X116A-X215F-X269R, X024R-X043R-X101A-X116A, X024R-X043R-X101A-X116A-X215F-X269R, X020R-X101G-X103A-X104I-X215F-X232V-X245R, X043R-X101A-X269R, X024R-X043R-X116A-X213A-X269R, X020R-X024R-X043R-X045T-X101A-X213A, X024R-X043R-X116A-X215F-X269R, X020R-X024R-X213A-X215F, X020R-X116A-X269R, X024R-X116A-X213A-X269R, X043R-X101A-X116A-X269R, X101G-X103A-X104I-X116A-X213A-X232V-X245R-X269R, X024R-X043R-X045T-X101A-X116A-X215F-X269R, X020R-X043R-X101A-X269R, X101A-X103A-X104I-X213A-X232V-X245R-X269R, X024R-X215F-X269R, X043R-X101A-X116A-X213A-X215F-X269R, X043R-X101A-X213A-X269R, X020R-X024R-X043R-X045T-X116A-X213A, X101G-X103A-X104I-X232V-X245R-X269R, X024R-X043R-X045T-X101A-X116A-X213A-X269R, X024R-X043R-X045T-X269R, X020R-X043R-X045T-X101A-X269R, X024R-X043R-X116A-X269R, X020R-X024R-X043R-X045T, X043R-X116A-X269R, X024R-X043R-X101A-X215F-X269R, X024R-X043R-X045T-X213A-X215F-X269R, X020R-X024R-X045T-X269R, X020R-X043R-X101A-X116A-X213A-X215F, X020R-X101G-X103A-X104I-X213A-X215F-X232V-X245R, X020R-X024R-X045T-X116A-X269R, X020R-X101A-X116A-X269R, X024R-X043R-X215F, X020R-X024R-X213A, X024R-X043R-X101A-X215F, X020R-X024R-X043R-X045T-X116A, X020R-X024R-X043R-X045T-X101A-X269R, X020R-X024R-X101A-X215F, X020R-X024R-X116A-X213A-X215F, X020R-X024R-X116A, X020R-X024R-X101A-X116A, X043R-X213A-X215F-X269R, X024R-X101A-X269R, X024R-X043R-X116A-X215F, X020R-X038A-X043R-X101A, X020R-X024R-X116A-X215F, X024R-X043R-X101A-X213A, X014L-X020R-X024R-X043R-X045T-X101A-X215F, X020R-X024R-X215F, X020R-X116A-X215F-X269R, X020R-X045T-X116A-X269R, X020R-X024R-X043R-X045T-X215F, and X020R-X024R-X043R-X045T-X116A-X213A-X215F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
22 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X043R-X076D-X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R-X271F, X024R-X043R-X076D-X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R-X271F-X271F, X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R-X271F, X076D-X101G-X103A-X104I-X114V-X158E-X188D-X232V-X245R-X248D-X249R-X271F, X024R-X076D-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R-X271F, X024R-X043R-X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X024R-X043R-X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101A-X103A-X104I-X158E-X166D-X188D-X217E-X232V-X245R-X248D-X249R, X076D-X101G-X103A-X104I-X128L-X158E-X188D-X232V-X245R-X248D-X249R-X271F, and X043R-X076D-X101G-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
23 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X022A-X101G-X103A-X104I-X159D-X217E-X232V-X245R-X248D-X271F, X022A-X043R-X101G-X103A-X104I-X159D-X188D-X217E-X232V-X245R-X248D-X271F, X022A-X101G-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X271F, X043R-X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X043R-X076D-X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R-X271F, X024R-X101G-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X022A-X076D-X101G-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X271F, X022A-X043R-X076D-X101G-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X271F, X022A-X076D-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, X022A-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, X076D-X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R-X271F, X043R-X101A-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R, X024R-X076D-X101A-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, X076D-X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101A-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, X043R-X076D-X101A-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, X101G-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R-X271F, X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X076D-X101A-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, X101G-X103A-X104I-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128L-X158E-X188D-X232V-X245R-X248D-X249R, X076D-X101G-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R-X271F, X043R-X076D-X101A-X103A-X104I-X158E-X166D-X188D-X232V-X245R-X248D-X249R, and X076D-X101A-X103A-X104I-X158E-X188D-X217E-X232V-X245R-X248D-X249R-X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
24 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X017R-X022A-X076D-X101G-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X271F, X022A-X043R-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, X022A-X101G-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X249R-X271F, X017R-X022A-X076D-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, X022A-X076D-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X249R-X271F, X022A-X101G-X102A-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X271F, and X022A-X043R-X076D-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X249R-X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
25 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101S-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101S-X103G-X104V-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103S-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101A-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101A-X103A-X104L-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103G-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101S-X103G-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101S-X103S-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101S-X103S-X104V-X158E-X188D-X232V-X245R-X248D-X249R, X101A-X103S-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101A-X103S-X104I-X159E-X232V-X245R-X248D-X249R, X101S-X103A-X104I-X159E-X232V-X245R-X248D-X249R, X101G-X103A-X104L-X158E-X188D-X232V-X245R-X248D-X249R, X101A-X103A-X104L-X159E-X232V-X245R-X248D-X249R, X101A-X103S-X104L-X159E-X232V-X245R-X248D-X249R, X101G-X103S-X104L-X159E-X232V-X245R-X248D-X249R, X101S-X103A-X104L-X159E-X232V-X245R-X248D-X249R, X101A-X103G-X104V-X159E-X232V-X245R-X248D-X249R, and X101S-X103A-X104V-X159E-X232V-X245R-X248D-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
26 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X026F-X051W-X104L-X106E, X026F-X031F-X078N-X102A-X160D, X020K-X100S-X116L-X158E-X166D-X243F, X033S-X043W-X218D-X239G-X243F, X022L-X038F-X048R-X062E-X100S-X186K, X101D-X103N-X116L-X144R-X215D, X104L-X105T-X213A-X217E-X256N, X043W-X101D-X212M-X243F, X026F-X048R-X105T-X213A-X218D-X224A, X024F-X101D-X118R-X215D-X250I-X272F, X121F-X185E-X224A-X239G, X022L-X031F-X102A-X128D-X224A-X243F, X062E-X078N-X102A-X116L-X144R-X250I, X022L-X038F-X121F-X160D-X272F, X026F-X078N-X159C-X186K-X243F, X024F-X048R-X118R-X166D-X217E, X023A-X038F-X078N-X100S-X212M-X215D, X100S-X116L-X158E-X213A, X078N-X104L-X118R-X128D, X102A-X103N-X105T-X194E, X022L-X078N-X128D-X213A, X027R-X100S-X118R-X160D-X188D-X243F, X024F-X102A-X186K-X213A-X217E-X243F, X033S-X105T-X188D-X216F, X023A-X100S-X194E-X212M, X048R-X128D-X185E-X239G, X020K-X024F-X033S-X129E-X194E, X020K-X027R-X129E-X166D-X239G, X022L-X023A-X027R-X101D-X104L-X216F, X033S-X118R-X129E-X194E-X239G, X022L-X078N-X116L-X129E-X256N, X027R-X101D-X103N-X105T-X272F, X048R-X078N-X116L-X185E-X217E-X239G, X023A-X024F-X027R-X062E, X024F-X103N-X104L-X118R-X188D, X026F-X104L-X256N-X272F, X024F-X043W-X104L-X121F-X129E, X062E-X078N-X116L-X224A, X023A-X024F-X051W-X158E, X027R-X038F-X102A-X116L, X062E-X078N-X144R-X212M, X031F-X116L-X256N-X272F, X022L-X033S-X104L-X116L-X160D-X186K, X024F-X118R-X129E-X186K-X213A, X043W-X105T-X213A-X215D-X216F, X031F-X105T-X186K-X188D, X026F-X194E-X213A-X256N, and X103N-X160D-X250I-X256N, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
27 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X022A-X024R-X101D-X103A-X104I-X118R-X159D-X188D-X232V-X248D-X271F, X022A-X024R-X103A-X104I-X129E-X159D-X188D-X232V-X248D-X271F, X022A-X024R-X103A-X104I-X118R-X159D-X188D-X217D-X232V-X248D, X022A-X024R-X101D-X103A-X104I-X118R-X129E-X159D-X188D-X232V-X245R-X248D, X022A-X024R-X101D-X103A-X104I-X159D-X188D-X232V-X245R-X248D, X022A-X043R-X103A-X104I-X118R-X129E-X159D-X188D-X232V-X245R-X248D, X022A-X043R-X103A-X104I-X118R-X128I-X129E-X159D-X188D-X232V-X248D, X022A-X043R-X101D-X103A-X104I-X118R-X129E-X159D-X188D-X232V-X248D-X271F, X022A-X024R-X043R-X101D-X103A-X104I-X159D-X188D-X232V-X245R-X248D, X022A-X103A-X104I-X159D-X188D-X232V-X248D, X022A-X024R-X103A-X104I-X118R-X129E-X159D-X188D-X232V-X248D-X271F, X022A-X024R-X103A-X104I-X159D-X188D-X217D-X232V-X245R-X248D-X271F, X022A-X043R-X062E-X103A-X104I-X159D-X188D-X232V-X245R-X248D-X271F, X022A-X043R-X103A-X104I-X129E-X159D-X188D-X232V-X245R-X248D, X022A-X024R-X103A-X104I-X159D-X188D-X217D-X232V-X248D-X271F, X022A-X103A-X104I-X118R-X159D-X188D-X217D-X232V-X245R-X248D, X022A-X024R-X101D-X103A-X104I-X118R-X128I-X159D-X188D-X232V-X245R-X248D, X022A-X024R-X043R-X103A-X104I-X159D-X188D-X217D-X232V-X248D-X271F, X022A-X043R-X103A-X104I-X118R-X159D-X188D-X217D-X232V-X248D-X271F, X022A-X043R-X103A-X104I-X118R-X159D-X188D-X232V-X248D-X271F, X022A-X103A-X104I-X128I-X129E-X159D-X188D-X232V-X248D-X271F, X022A-X103A-X104I-X159D-X188D-X217D-X232V-X245R-X248D-X271F, X022A-X043R-X103A-X104I-X128I-X159D-X188D-X232V-X245R-X248D, X022A-X101D-X103A-X104I-X118R-X159D-X188D-X217D-X232V-X245R-X248D-X271F, X022A-X103A-X104I-X118R-X129E-X159D-X188D-X232V-X245R-X248D-X271F, X022A-X024R-X043R-X103A-X104I-X118R-X159D-X188D-X217D-X232V-X248D, X022A-X062E-X103A-X104I-X118R-X159D-X188D-X232V-X245R-X248D, X022A-X043R-X101D-X103A-X104I-X118R-X129E-X159D-X188D-X217D-X232V, X022A-X024R-X103A-X104I-X159D-X188D-X217D-X232V-X248D, X022A-X024R-X043R-X103A-X104I-X118R-X128I-X129E-X159D-X188D-X217D-X232V-X248D-X271F, and X022A-X103A-X104I-X118R-X159D-X188D-X217D-X232V-X245R-X248D-X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
28 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020K-X024F-X062E-X188D-X239G, X024F-X062E-X116L-X239G, X020K-X023A-X062E-X188D, X020K-X023A-X024F-X062E-X118R-X188D-X213A, X020K-X043W-X062E-X116L-X188D-X213A-X239G, X023A-X062E-X116L-X118R, X023A-X024F-X062E-X116L-X118R, X024F-X116L, X024F-X062E-X188D-X213A, X023A-X062E-X116L-X118R-X188D-X239G, X020K-X024F-X062E, X020K-X043W-X062E-X116L-X239G, X024F-X062E-X116L-X213A-X239G, X020K-X024F-X043W-X062E-X116L-X213A, X020K-X023A-X024F-X062E-X116L-X188D-X213A, X024F-X062E-X188D-X239G, X023A-X043W-X062E-X116L-X118R-X213A, X062E-X188D-X239G, X020K-X024F-X062E-X239G, X024F-X116L-X118R-X188D-X239G, X020K-X023A-X062E-X116L-X118R-X213A, X020K-X023A-X024F-X062E-X188D-X213A-X239G, X024F-X043W-X118R-X188D, X023A-X024F-X116L-X118R-X188D-X213A, X020K-X023A-X043W-X116L-X188D-X213A-X239G, X023A-X024F-X116L-X188D-X239G, X023A-X043W-X116L-X118R-X188D, X023A-X024F-X118R-X188D-X239G, X023A-X024F-X043W-X062E-X116L-X118R, X020K-X043W-X188D-X213A, X024F-X062E-X118R-X239G, X023A-X043W-X188D-X213A, X020K-X024F-X043W-X062E-X116L-X118R-X188D-X239G, X020K-X116L-X188D-X239G, X020K-X043W-X062E-X118R, X020K-X043W-X116L-X188D-X213A, X020K-X024F, X023A-X043W-X116L-X239G, X023A-X024F-X043W-X116L-X118R-X188D-X239G, X020K-X023A-X043W-X213A, and X023A-X024F-X062E-X118R-X213A-X239G, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
29 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020K-X023A-X043W-X118R-X128I-X129E-X159D-X188D, X024F-X118R-X128I-X129E-X159D, X020K-X024F-X062E-X116L-X118R-X188D, X020K-X062E-X116L-X188D, X062E-X116L-X118R-X213A, X020K-X023A-X062E-X116L-X188D, X062E-X116L-X118R-X188D, X020K-X062E-X116L-X213A, X020K-X023A-X062E-X116L, X020K-X062E-X188D-X213A, X020K-X062E, X020K-X024F-X062E-X116L-X188D, X020K-X043W-X062E-X116L-X188D, X020K-X024F-X062E-X188D-X213A, X062E-X116L-X188D-X213A, X020K-X062E-X116L, X020K-X023A-X062E-X116L-X188D-X213A, X023A-X024F-X062E-X116L-X213A, X022A-X043R-X103A-X104I-X128I-X129E-X159D-X188D-X232V-X245R-X248D, X022A-X043R-X103A-X104I-X118R-X128I-X129E-X159D-X188D-X232V-X248D-X271F, X024F-X062E-X116L-X188D, X022A-X024R-X103A-X104I-X118R-X128I-X129E-X159D-X188D-X232V-X248D, X023A-X062E-X116L-X188D, X043W-X062E-X116L, X020K-X023A-X116L-X188D, X043W-X062E-X116L-X188D, X024F-X062E-X116L, X062E-X116L-X188D, and X022A-X024R-X103A-X104I-X128I-X159D-X188D-X232V-X248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
30 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020K-X023A-X043W-X118R-X128I-X129E-X159D-X188D, X024F-X118R-X128I-X129E-X159D, X020K-X024F-X062E-X116L-X118R-X188D, X020K-X062E-X116L-X188D, X062E-X116L-X118R-X213A, X020K-X023A-X062E-X116L-X188D, X062E-X116L-X118R-X188D, X020K-X062E-X116L-X213A, X020K-X023A-X062E-X116L, X020K-X062E-X188D-X213A, X020K-X062E, X020K-X024F-X062E-X116L-X188D, X020K-X043W-X062E-X116L-X188D, X020K-X024F-X062E-X188D-X213A, X062E-X116L-X188D-X213A, X020K-X062E-X116L, X020K-X023A-X062E-X116L-X188D-X213A, X023A-X024F-X062E-X116L-X213A, X022A-X043R-X103A-X104I-X128I-X129E-X159D-X188D-X232V-X245R-X248D, X022A-X043R-X103A-X104I-X118R-X128I-X129E-X159D-X188D-X232V-X248D-X271F, X024F-X062E-X116L-X188D, X022A-X024R-X103A-X104I-X118R-X128I-X129E-X159D-X188D-X232V-X248D, X023A-X062E-X116L-X188D, X043W-X062E-X116L, X020K-X023A-X116L-X188D, X043W-X062E-X116L-X188D, X024F-X062E-X116L, X062E-X116L-X188D, and X022A-X024R-X103A-X104I-X128I-X159D-X188D-X232V-X248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
31 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X024R-X086W-X118R, X024R-X078R-X086W-X243F, X024R-X033S-X086S-X087N-X209A, X033S-X118R, X024R-X078R-X086W-X118R-X270T, X024R-X033S-X086W-X118R, X078R-X086W-X243F, X033S-X078R-X086W-X118R-X209A, X033S-X078R-X209A, X086W-X118R-X243F, X024R-X086W, X078R-X086W-X235F, X024R-X118R, X024R-X086R, X101G-X103A-X104I-X232V, X024R-X033S-X078R-X086W-X118R, X024R-X118R-X209A, X209A-X241R, X033S-X086W-X243F, X033S-X172V-X209A, X118R-X209A-X243F, X024R-X086S-X141G, X024R-X118R-X209A-X243F, X024R-X033S-X086S-X085N-X235F, X024R-X033S-X133V, X024R-X033S-X078R-X086W, X024R-X086W-X209A, X024R-X241R, X033S-X118R-X243F, X024R-X235F, X024R-X078R-X086W, X024R-X118R-X209A-X235F, X024R-X209A-X241R, X033S-X118R-X241R, X086W-X118R-X209A, X033S-X118R-X159D-X209A, X033S-X078R-X086W, X024R-X086W-X243F, X118R-X209A, X024R-X086W-X118R-X203I, X078R-X209A-X235F, X024R-X033S-X241R, X078R-X118R, X033S-X118R-X209A-X243F, X021M-X024R-X033S, X024R-X033S-X086W, X033S-X235F, X078R-X086W-X209A, X024R-X033S-X209A-X235F, X033S-X086W-X118R, X024R-X033S-X078R-X209A, X033S-X086W-X118R-X209A-X243F, X086W-X209A-X243F, X005S-X078R-X118R-X241R, X024R-X174T, X033S-X209A-X243F, X086W-X118R-X133V, X024R-X033S-X118R, X024R-X086W-X209A-X235F, X086W-X209A, X008T-X024R, X086W-X118R, X033S-X241R, X005S-X024R-X033S-X243F, X024R-X209A-X242P, X024R-X033S-X078R-X118R, X024R-X033S-X194T, X024R-X243F, X024R-X209A, X024R-X033S-X118R-X209A, X033S-X086W, X024R-X033S, X024R-X033S-X078R-X243F, X086W-X243F, X033S-X118D-X138V-X209A, X033S-X209A-X235F, X024R-X086R-X118R, X033S-X201S, X024R-X239Q, X033S-X118R-X209A-, X078R-X086W, X235F-X243F, X024R-X209A-X235F, X118R-X172V, X017Y-X024R-X033S-X086W, X033S-X148F, X024R-X118R-X235F, X033S-X078R, X033S-X243F, X024C-X033S, X118R-X194T, X033S-X209A, X118R-X209A-X235F, X024R-X033S-X209A-X243F, X024R-X033S-X235F, X024R-X033S-X118R-X235F, X024R-X141G, X024R-X274I, X024R-X033S-X209A, X086W-X235F, X024R-X209A-X243F, X004E-X033S-X078R, X086W-X209A-X235F, X015T-X033S, X033S-X086W-X156L-X209A, X024R-X118R-X243F-X269H, X209A-X235F, X024R-X247H, X024R-X033S-X228T, X078R-X235F, X024R-X033S-X174V-X235F, X024R-X235F-X243F, X024R-X033S-X235F-X241R, X024R-X033S-X151V, X024R-X104A, X033S-X048T, X012H-X104A-X118R, X118R-X235F, X033S-X253A, X143A-X209A, X024R-X033S-X243F, X033S-X239T, X209A-X243F, X024R-X033S-X129H-X184D-X253M, X024R-X085V-X086W-X118R-X235F, X024R-X272P, and X024R-X269C, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
32 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X020R-X087D-X101G-X103A-X104I-X232V-X245R, X020R-X101G-X103A-X104I-X150L-X232V-X245R, X018R-X020R-X024R-X076D-X087D-X249R, X018R-X020R-X024R-X076D-X150L-X249R, X018R-X024R-X043R-X076D-X087D-X249R, X018R-X024R-X043R-X076D-X150L-X249R, X018R-X024R-X076D-X078R-X087D-X249R, X018R-X024R-X076D-X078R-X150L-X249R, X018R-X024R-X076D-X087D-X249R-X269R, X018R-X024R-X076D-X087D-X242R-X249R, X018R-X024R-X076D-X087D-X150L-X249R, X018R-X024R-X076D-X150L-X249R, X018R-X087D-X101G-X103A-X104I-X232V-X245R, X018R-X101G-X103A-X104I-X150L-X232V-X245R, X018R-X022R-X024R-X076D-X087D-X249R, X018R-X022R-X024R-X076D-X150L-X249R, X043R-X087D-X101G-X103A-X104I-X232V-X245R-X269R, X043R-X101G-X103A-X104I-X150L-X232V-X245R, X024R-X087D-X101G-X103A-X104I-X232V-X245R, X024R-X101G-X103A-X104I-X150L-X232V-X245R, X078R-X087D-X101G-X103A-X104I-X232V-X245R, X078R-X101G-X103A-X104I-X150L-X232V-X245R, X087D-X101G-X103A-X104I-X232V-X245R-X269R, X101G-X103A-X104I-X150L-X232V-X245R-X249R, X101G-X103A-X104I-X150L-X232V-X245R-X269R, X022R-X087D-X101G-X103A-X104I-X232V-X245R, X018R-X024R-X043D-X076D-X150L-X249R, X043R-X087D-X101G-X103A-X104I-X232V-X245R, X022R-X101G-X103A-X104I-X150L-X232V-X245R, X018R-X024R-X043D-X076D-X087D-X249R, X018R-X024R-X076D-X087D-X249R, X018R-X024R-X076D-X150L-X242R-X249R, X043R-X101G-X103A-X104I-X150L-X232V-X245R-X269R, X076D-X101G-X103A-X104I-X150L-X232V-X245R, X087D-X101G-X103A-X104I-X232V-X242R-X245R, X101G-X103A-X104I-X150L-X232V-X245R, X076D-X087D-X101G-X103A-X104I-X232V-X245R, X087D-X101G-X103A-X104I-X232V-X245R, and X101G-X103A-X104I-X150L-X232V-X242R-X245R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
33 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X024R-X101G-X103A-X104I-X129Q-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X130A-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129Q-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129Q-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129Q-X130A-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128L-X129Q-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129Q-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129Q-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129Q-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128L-X129Q-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X128L-X129Q-X158E-X188D-X232V-X245R-X248D-X249R, and X024R-X027R-X101G-X103A-X104I-X128L-X129Q-X130A-X158E-X188D-X232V-X245R-X248D-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
34 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101G-X103A-X104I-X232V-X222Q-X245R, X101G-X103A-X104I-X158E-X188D-X222S-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X158E-X188D-X222Q-X232V-X245R-X248D-X249R, X076D-X101G-X103A-X104I-X232V-X222Q-X245R, X101G-X103A-X104I-X232V-X222S-X245R, X076D-X101G-X103A-X104I-X232V-X222S-X245R, and X076D-X101G-X103A-X104I-X158E-X188D-X222S-X232V-X245R-X248D-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
35 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X024R-X101G-X103A-X104I-X128L-X129Q-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X128L-X129Q-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129Q-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X130A-X158E-X183D-X188D-X217E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128L-X129Q-X130A-X158E-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X129Q-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X128L-X130A-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128L-X129Q-X158E-X183D-X188D-X232V-X245R-X248D-X249R, I03A-X104I-X128L-X129Q-X130A-X158E-X183D-X188D-X024R-X101G-X103A-X104I-X128L-X129Q-X158E-X188D-X232V-X245R-X248D-X249R-X271G, X101G-X103A-X104I-X129Q-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X130A-X158E-X188D-X217E-X232V-X245R-X248D-X249R, X024R-X101G-X103A-X104I-X128L-X158E-X183D-X188D-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X129Q-X158E-X188D-X217E-X232V-X245R-X248D-X249R, and X024R-X101G-X103A-X104I-X128L-X130A-X158E-X188D-X232V-X245R-X248D-X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
36 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X1R, X2W, X2M, X2R, X2A, X2S, X3R, X4R, X4C, X4S, X8A, X9F, X9W, X9A, X10S, X10M, X10H, X10A, X12R, X12F, X14K, X14F, X14Q, X15R, X15F, X16S, X17R, X17M, X17F, X18R, X18K, X20F, X20R, X20K, X22Y, X22A, X22R, X22V, X22Q, X22W, X22L, X23F, X23S, X23A, X24W, X24R, X24H, X24F, X24Q, X24L, X25V, X25F, X25R, X26F, X27V, X27F, X27L, X27R, X28N, X28E, X28A, X29T, X30E, X31F, X33D, X33G, X33S, X34P, X35M, X36F, X36R, X36T, X38L, X38F, X38R, X40L, X40W, X40N, X40R, X40T, X40H, X42I, X43D, X43I, X43R, X43M, X43F, X43W, X43S, X43A, X45T, X46R, X48R, X50C, X51H, X51W, X51F, X52F, X52E, X52N, X55Y, X57R, X59R, X59A, X59F, X60A, X60Q, X60P, X62E, X62Q, X63I, X63V, X63T, X63P, X63D, X63M, X63H, X63Q, X63E, X63A, X63S, X64F, X64T, X68C, X68A, X69N, X69P, X69W, X69T, X71G, X72C, X74C, X75F, X75A, X75R, X75E, X76D, X78I, X78R, X78N, X79W, X79Q, X81R, X82V, X82T, X82F, X82M, X82R, X85M, X86L, X86I, X86W, X89P, X89T, X89V, X89G, X89W, X89H, X89F, X89L, X89I, X91N, X91F, X92F, X94N, X99G, X99F, X99M, X99T, X99P, X100I, X100S, X100N, X100Q, X101N, X101A, X101G, X101P, X101F, X101E, X101T, X101D, X102H, X102N, X102E, X102T, X102A, X103G, X103D, X103N, X104D, X104E, X104I, X104L, X105Q, X105E, X105T, X106F, X106V, X106G, X106E, X106T, X106D, X106A, X107F, X107M, X108G, X108I, X109M, X111V, X111I, X112V, X112L, X112Q, X114G, X115K, X115R, X116A, X116K, X116L, X117F, X118I, X118R, X119C, X120F, X120A, X120R, X121E, X121F, X123G, X123E, X124S, X128F, X128H, X128I, X128L, X128Q, X128N, X128M, X128D, X129E, X132E, X132A, X138G, X144R, X147L, X148I, X158E, X158E, X159E, X159C, X160D, X166D, X166E, X167W, X175V, X177C, X181A, X182R, X183F, X183I, X183D, X183R, X183M, X185E, X185I, X185V, X186H, X186K, X188R, X188E, X188D, X192H, X192W, X194V, X194F, X194E, X197F, X198L, X198F, X203E, X203C, X208S, X209N, X209F, X209E, X209S, X209H, X209G, X209T, X209L, X210R, X210V, X210L, X211R, X211Q, X212I, X212M, X212F, X213A, X214F, X215F, X215N, X215D, X215H, X215E, X216F, X216A, X217N, X217E, X217D, X218P, X218D, X218E, X224A, X224G, X227I, X230E, X231I, X231C, X233C, X234F, X235F, X236F, X236N, X238L, X238K, X238R, X239K, X239S, X239T, X239G, X239H, X239R, X239N, X239F, X240R, X241R, X242L, X242R, X243R, X243F, X244R, X246S, X248I, X248V, X248R, X249R, X249T, X250I, X251S, X251R, X252I, X252F, X252H, X252R, X253F, X253I, X253R, X254C, X256N, X258R, X260V, X260I, X262H, X262D, X263F, X265F, X267N, X267V, X267M, X269I, X269R, X270C, X271F, X271V, X271I, X271P, X271H, X271M, X271T, X271L, X271A, X272F, X272R, X272F, X273I, X273F, and X274G, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
37 . An isolated subtilisin variant of claim 35 , wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X16S, X18R, X20R, X22A, X24R, X43R/D, X45T, X76D, X101A, X103G, X104L, X111V, X128N, X148I, X230E, X242R, and X249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
38 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X1R, X230E, X271L, X115R, X20R, X249R, X235F, X27V/F/L, X75E, X82R, X18R, X269R, X43D, X43R, X76D, X45T, X212F, X242R, X24R, X78R, X9A, X22R, X121E, X244R, X28E, X30E, X4R, and X241R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
39 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X062E-X158E, X103G-X158E, X128N-X158E, X016S-X158E, X104L-X158E, X089P-X158E, X111V-X158E, X022A-X158E, X101A-X158E, X148I-X158E, X129E-X158E, X022A-X089P, X016S-X089P, X062E-X089P, X062E-X271F, X158E-X271F, X186H-X271F, X129E-X271F, X111V-X271F, X209E-X271F, X016S-X271F, X188D-X271F, X022A-X271F, X159E-X271F, X104L-X271F, X101A-X271F, X089P-X271F, X128N-X271F, X103G-X271F, X148I-X271F, X249R-X271F, X062E-X159E, X016S-X159E, X128N-X159E, X148I-X159E, X111V-X159E, X089P-X159E, X022A-X159E, X129E-X159E, X103G-X159E, X104L-X159E, X158E-X159E, X101A-X159E, X158E-X249R, X111V-X249R, X129E-X249R, X062E-X249R, X016S-X249R, X186H-X249R, X148I-X249R, X159E-X249R, X101A-X249R, X188D-X249R, X104L-X249R, X209E-X249R, X022A-X249R, X128N-X249R, X103G-X249R, X089P-X249R, X022A-X111V, X101A-X111V, X016S-X111V, X104L-X111V, X062E-X111V, X103G-X111V, X089P-X111V, X016S-X148I, X062E-X148I, X022A-X148I, X129E-X148I, X104L-X148I, X103G-X148I, X128N-X148I, X101A-X148I, X089P-X148I, X111V-X148I, X016S-X062E, X022A-X062E, X062E-X129E, X022A-X129E, X128N-X129E, X016S-X129E, X101A-X129E, X104L-X129E, X089P-X129E, X103G-X129E, X111V-X129E, X062E-X186H, X128N-X186H, X101A-X186H, X022A-X186H, X016S-X186H, X158E-X186H, X089P-X186H, X129E-X186H, X159E-X186H, X103G-X186H, X104L-X186H, X111V-X186H, X148I-X186H, X062E-X101A, X022A-X101A, X016S-X101A, X089P-X101A, X062E-X103G, X022A-X103G, X016S-X103G, X101A-X103G, X089P-X103G, X062E-X128N, X016S-X128N, X022A-X128N, X101A-X128N, X104L-X128N, X089P-X128N, X103G-X128N, X111V-X128N, X111V-X188D, X062E-X188D, X016S-X188D, X148I-X188D, X022A-X188D, X128N-X188D, X101A-X188D, X104L-X188D, X089P-X188D, X129E-X188D, X159E-X188D, X186H-X188D, X103G-X188D, X158E-X188D, X016S-X022A, X016S-X104L, X022A-X104L, X101A-X104L, X062E-X104L, X103G-X104L, X089P-X104L, X159E-X209E, X111V-X209E, X101A-X209E, X016S-X209E, X128N-X209E, X148I-X209E, X129E-X209E, X062E-X209E, X022A-X209E, X103G-X209E, X158E-X209E, X188D-X209E, X104L-X209E, X089P-X209E, and X186H-X209E, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
40 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X018R-X241R, X020R-X241R, X024R-X241R, X009A-X241R, X020R-X241R, X004R-X241R, X043R-X241R, X078R-X241R, X022R-X241R, X115R-X241R, X001R-X241R, X212F-X241R, X082R-X241R, X018R-X244R, X024R-X244R, X078R-X244R, X020R-X244R, X212F-X244R, X009A-X244R, X082R-X244R, X001R-X244R, X043R-X244R, X022R-X244R, X004R-X244R, X115R-X244R, X241R-X244R, X242R-X244R, X001R-X004R, X009A-X022R, X018R-X022R, X020R-X022R, X004R-X022R, X001R-X022R, X024R-X242R, X018R-X242R, X004R-X242R, X020R-X242R, X212F-X242R, X082R-X242R, X078R-X242R, X001R-X242R, X009A-X242R, X022R-X242R, X115R-X242R, X043R-X242R, X241R-X242R, X018R-X212F, X022R-X212F, X004R-X212F, X024R-X212F, X001R-X212F, X115R-X212F, X020R-X212F, X009A-X212F, X043R-X212F, X078R-X212F, X082R-X212F, X009A-X078R, X020R-X078R, X024R-X078R, X022R-X078R, X018R-X078R, X004R-X078R, X001R-X078R, X043R-X078R, X022R-X024R, X020R-X024R, X018R-X024R, X001R-X024R, X004R-X024R, X009A-X024R, X004R-X009A, X001R-X009A, X242R-X269R, X024R-X269R, X020R-X269R, X022R-X269R, X249R-X269R, X212F-X269R, X043R-X269R, X244R-X269R, X001R-X269R, X018R-X269R, X078R-X269R, X009A-X269R, X115R-X269R, X241R-X269R, X004R-X269R, X082R-X269R, X018R-X043R, X020R-X043R, X004R-X043R, X022R-X043R, X009A-X043R, X001R-X043R, X024R-X043R, X009A-X018R, X004R-X018R, X001R-X018R, X024R-X082R, X009A-X082R, X018R-X082R, X001R-X082R, X078R-X082R, X020R-X082R, X022R-X082R, X004R-X082R, X043R-X082R, X043R-X249R, X020R-X249R, X004R-X249R, X018R-X249R, X009A-X249R, X212F-X249R, X022R-X249R, X024R-X249R, X115R-X249R, X001R-X249R, X082R-X249R, X242R-X249R, X241R-X249R, X244R-X249R, X078R-X249R, X018R-X115R, X020R-X115R, X022R-X115R, X078R-X115R, X009A-X115R, X004R-X115R, X001R-X115R, X082R-X115R, X043R-X115R, X024R-X115R, X009A-X020R, X018R-X020R, X004R-X020R, X001R-X020R, X009A-X271L, X020R-X271L, X024R-X271L, X244R-X271L, X241R-X271L, X043R-X271L, X022R-X271L, X249R-X271L, X212F-X271L, X115R-X271L, X242R-X271L, X078R-X271L, X004R-X271L, X269R-X271L, X001R-X271L, X018R-X271L, and X082R-X271L, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
41 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101G-X103A-X104I, X22A-X101A-X209E, S103G-L111V-G159E, X22A-X103G-X159E, X22A-X111V-X159E, X22A-X128N-X271F-X209E, X22A-X103G-X111V, X62E-X111V-X128N, X22A-X111V-X128N, X22A-X62E-X111V, X101A-X103G-X104L-X188D, X101G-X103A-X104I-X159D, X101A-X103G-X104L-X128N, X22A-X101A-X159E, X101A-X103G-X104L, X101A-X103G-X104L-X159E, X22A-X101A-X103G-X104L, X101A-X103G-X104L-X209E, X22A-X209E-X271F, X22A-X101A-X271F, and X101A-X209E-X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
42 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X103A-X104I-X159D-X232V-X236H-X245R-X248D-X252K, X101G-X104I-X159D-X232V-X236H-X245R-X248D-X252K, X101G-X103A-X159D-X232V-X236H-X245R-X248D-X252K, X101G-X103A-X104L-X232V-X236H-X245R-X248D-X252K, X101G-X103A-X104L-X159D-X236H-X245R-X248D-X252K, X101G-X103A-X104L-X159D-X232V-X245R-X248D-X252K, X101G-X103A-X104L-X159D-X232V-X236H-X248D-X252K, X101G-X103A-X104L-X159D-X232V-X236H-X245R-X252K, and X101G-X103A-X104L-X159D-X232V-X236H-X245R-X248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
43 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101G-X103A-X104I-X159D-X232V-X245R-X248D, X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, X101G-X103A-X104I-X159D-X232V-X245R-X248D-X238R, X101G-X103A-X104I-X159D-X232V-X245R-X248D-X248R, X101G-X103A-X104I-X159D-X232V-X245R-X248D-X253R, X101G-X103A-X104I-X159D-X232V-X245R-X248D-X24R, X101G-X103A-X104I-X159D-X232V-X245R-X248D-X76D, X101G-X103A-X104I-X159E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X159E-X232V-X245R-X248D-X271F, X101G-X103A-X104I-X158E-X232V-X245R-X248D-X249R, X101G-X103A-X104I-X158E-X232V-X245R-X248D-X271F, X22A-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X249R, X22A-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, X62E-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X249R, and X62E-X101G-X103A-X104I-X159D-X232V-X245R-X248D-X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
44 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X20R-X43R-X249R, X20R-X22R-X43R, X20R-X43R-X242R, X20R-X43R-X271L, X20R-X43R-X244R, X20R-X24R-X43R-X242R, X9A-X22R-X78R-X212F-X241R, X9A-X20R-X43R-X212F, X9A-X43R-X212F, X20R-X43R-X212F, X20R-X22R-X43R-X212F, X24R-X78R-X212F, X9A-X43R-X78R, X9A-X43R-X78R-X242R, X9A-X20R-X43R-X78R, X20R-X24R-X43R-X78R-X242R, X22R-X24R-X78R-X212F, X9A-X20R-X43R-X78R-X242R, X20R-X43R-X78R-X249R, X20R-X43R-X78R, X9A-X78R-X212F, X9A-X22R-X43R-X78R, X9A-X20R-X24R-X43R, X9A-X22R-X78R-X212F, X4R-X9A-X22R-X78R-X212F, X20R-X24R-X43R, X1R-X9A-X43R, X20R-X24R-X43R-X115R, X9A-X24R-X43R, X20R-X22R-X24R-X43R, X1R-X24R-X43R, X9A-X20R-X24R-X43R-X242R, X9A-X20R-X22R-X78R-X212F, X9A-X24R-X43R-X244R, X9A-X24R-X43R-X242R, X4R-X9A-X22R-X24R-X212F, and X22R-X24R-X43R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
45 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X101G-X103A-X104I-X232V-X245R-X248D, X101G-X103A-X104I-X159D-X232V-X245R, X101G-X103A-X104I-X159R-X232V-X245R-X248D, X101G-X103A-X104I-X159D-X232V-X245R-X248R, X101G-X103A-X104I-X232V-X245R, X101G-X103A-X104I-X232V-X245R-X248R, X101G-X103A-X104I-X159R-X232V-X245R-X248R, and X101G, X103A, X104I, X232V, X236H, X245R, and X252K, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
46 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: X16S, X22A, X24R, X62E, X76D, X89P, X101A/G, X103G/A, X104L/I, X111V, X128N, X129E, X232V, X148I, X158E, X159D/E, X166D, X186H, X188D, X209E, X236H, X238R, X245R, X248D/R, X249R, X252K/R, X253R, and X271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
47 . An isolated subtilisin variant, wherein said subtilisin variant is a mature form having proteolytic activity and comprises an amino acid sequence comprising a combination of amino acid substitutions selected from: N062E-A158E, S103G-A158E, S128N-A158E, A016S-A158E, V104L-A158E, E089P-A158E, L111V-A158E, T022A-A158E, S101A-A158E, L148I-A158E, P129E-A158E, T022A-E089P, A016S-E089P, N062E-E089P, N062E-E271F, A158E-E271F, R186H-E271F, P129E-E271F, L111V-E271F, Y209E-E271F, A016S-E271F, S188D-E271F, T022A-E271F, G159E-E271F, V104L-E271F, S101A-E271F, E089P-E271F, S128N-E271F, S103G-E271F, L148I-E271F, H249R-E271F, N062E-G159E, A016S-G159E, S128N-G159E, L148I-G159E, L111V-G159E, E089P-G159E, T022A-G159E, P129E-G159E, S103G-G159E, V104L-G159E, A158E-G159E, S101A-G159E, A158E-H249R, L111V-H249R, P129E-H249R, N062E-H249R, A016S-H249R, R186H-H249R, L148I-H249R, G159E-H249R, S101A-H249R, S188D-H249R, V104L-H249R, Y209E-H249R, T022A-H249R, S128N-H249R, S103G-H249R, E089P-H249R, T022A-L111V, S101A-L111V, A016S-L111V, V104L-L111V, N062E-L111V, S103G-L111V, E089P-L111V, A016S-L148I, N062E-L148I, T022A-L148I, P129E-L148I, V104L-L148I, S103G-L148I, S128N-L148I, S101A-L148I, E089P-L148I, L111V-L148I, A016S-N062E, T022A-N062E, N062E-P129E, T022A-P129E, S128N-P129E, A016S-P129E, S101A-P129E, V104L-P129E, E089P-P129E, S103G-P129E, L111V-P129E, N062E-R186H, S128N-R186H, S101A-R186H, T022A-R186H, A016S-R186H, A158E-R186H, E089P-R186H, P129E-R186H, G159E-R186H, S103G-R186H, V104L-R186H, L111V-R186H, L148I-R186H, N062E-S101A, T022A-S101A, A016S-S101A, E089P-S101A, N062E-S103G, T022A-S103G, A016S-S103G, S101A-S103G, E089P-S103G, N062E-S128N, A016S-S128N, T022A-S128N, S101A-S128N, V104L-S128N, E089P-S128N, S103G-S128N, L111V-S128N, L111V-S188D, N062E-S188D, A016S-S188D, L148I-S188D, I022A-S188D, S128N-S188D, S101A-S188D, V104L-S188D, E089P-S188D, P129E-S188D, G159E-S188D, R186H-S188D, S103G-S188D, A158E-S188D, A016S-T022A, A016S-V104L, T022A-V104L, S101A-V104L, N062E-V104L, S103G-V104L, E089P-V104L, G159E-Y209E, L111V-Y209E, S101A-Y209E, A016S-Y209E, S128N-Y209E, L148I-Y209E, P129E-Y209E, N062E-Y209E, T022A-Y209E, S103G-Y209E, A158E-Y209E, S188D-Y209E, V104L-Y209E, E089P-Y209E, and R186H-Y209E, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
48 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: N018R-W241R, G020R-W241R, S024R-W241R, S009A-W241R, G020R-W241R, V004R-W241R, N043R-W241R, S078R-W241R, T022R-W241R, G115R-W241R, A001R-W241R, S212F-W241R, L082R-W241R, N018R-S024R-V244R, S078R-V244R, G020R-V244R, S212F-V244R, S009A-V244R, L082R-V244R, A001R-V244R, N043R-V244R, T022R-V244R, V004R-V244R, G115R-V244R, W241R-V244R, S242R-V244R, A001R-V004R, S009A-T022R, N018R-T022R, G020R-T022R, V004R-T022R, A001R-T022R, S024R-S242R, N018R-S242R, V004R-S242R, G020R-S242R, S212F-S242R, L082R-S242R, S078R-S242R, A001R-S242R, S009A-S242R, T022R-S242R, G115R-S242R, N043R-S242R, W241R-S242R, N018R-S212F, T022R-S212F, V004R-S212F, S024R-S212F, A001R-S212F, G115R-S212F, G020R-S212F, S009A-S212F, N043R-S212F, S078R-S212F, L082R-S212F, S009A-S078R, G020R-S078R, S024R-S078R, T022R-S078R, N018R-S078R, V004R-S078R, A001R-S078R, N043R-S078R, T022R-S024R, G020R-S024R, N018R-S024R, A001R-S024R, V004R-S024R, S009A-S024R, V004R-S009A, A001R-S009A, S242R-N269R, S024R-N269R, G020R-N269R, T022R-N269R, H249R-N269R, S212F-N269R, N043R-N269R, V244R-N269R, A001R-N269R, N018R-N269R, S078R-N269R, S009A-N269R, G115R-N269R, W241R-N269R, V004R-N269R, L082R-N269R, N018R-N043R, G020R-N043R, V004R-N043R, T022R-N043R, S009A-N043R, A001R-N043R, S024R-N043R, S009A-N018R, V004R-N018R, A001R-N018R, S024R-L082R, S009A-L082R, N018R-L082R, A001R-L082R, S078R-L082R, G020R-L082R, T022R-L082R, V004R-L082R, N043R-L082R, N043R-H249R, G020R-H249R, V004R-H249R, N018R-H249R, S009A-H249R, S212F-H249R, T022R-H249R, S024R-H249R, G115R-H249R, A001R-H249R, L082R-H249R, S242R-H249R, W241R-H249R, V244R-H249R, S078R-H249R, N018R-G115R, G020R-G115R, T022R-G115R, S078R-G115R, S009A-G115R, V004R-G115R, A001R-G115R, L082R-G115R, N043R-G115R, S024R-G115R, S009A-G020R, N018R-G020R, V004R-G020R, A001R-G020R, S009A-E271L, G020R-E271L, S024R-E271L, V244R-E271L, W241R-E271L, N043R-E271L, T022R-E271L, H249R-E271L, S212F-E271L, G115R-E271L, S242R-E271L, S078R-E271L, V004R-E271L, N269R-E271L, A001R-E271L, N018R-E271L, and L082R-E271L, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
49 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-N043R, G020K-N062E, S024F-N116L, G020K-S024F, S024R-A174T, S024R-G118R, S024R-K235F, S024R-P086R, S024R-P086W, S078R-G118R, T033S-G118R, T033S-K235F, Y209A-W241R, G020R-N076D, N018R-Q245R, S024R-R045T, A232V-Q245R, G118R-A172V, G118R-A194T, I008T-S024R, K235F-N243F, N018R-S103A, N018R-V104I, P086W-G118R, P086W-N243F, P086W-Y209A, S024C-T033S, S024R-A232V, S024R-N243F, S024R-P239Q, S024R-S101G, S024R-S141G, S024R-T033S, S024R-T274I, S024R-Y209A, S078R-P086W, S101G-A232V, T033S-L148F, T033S-P086W, T033S-P201S, T033S-S078R, T033S-W241R, T033S-Y209A, A230E-H249R, A232V-H249R, G118R-K235F, N076D-Q245R, P086W-K235F, S024R-R247H, S024R-V104A, S078R-K235F, S101G-H249R, S103A-A232V, T033S-A048T, T033S-P239T, T033S-T253A, T143A-Y209A, Y209A-K235F, N018R-R045T, Y209A-N243F, S024R-A272P, S024R-R269C, S101G-V104I, V104I-A232V, N076D-H249R, and S024R-N076D wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
50 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-N076D, S024R-R045T, A230E-H249R, N018R-R045T, N018R-Q245R, S101G-A232V, S024R-A232V, A232V-Q245R, S024R-S101G, N018R-V104I, N018R-S103A, S101G-H249R, A232V-H249R, S103A-A232V, N076D-Q245R, S101G-V104I, V104I-A232V, N076D-H249R, S024R-N076D, S024F-N116L, G020K-S024F, G020K-N062E, T033S-G118R, S024R-P086W, S024R-G118R, S024R-P086R, Y209A-W241R, S024R-W241R, S024R-K235F, G118R-Y209A, S078R-G118R, T033S-K235F, S024R-A174T, P086W-Y209A, I008T-S024R, P086W-G118R, T033S-W241R, S024R-N243F, S024R-Y209A, T033S-P086W, S024R-T033S, P086W-N243F, T033S-P201S, S024R-P239Q, S078R-P086W, K235F-N243F, G118R-A172V, T033S-L148F, T033S-S078R, T033S-N243F, S024C-T033S, G118R-A194T, T033S-Y209A, S024R-S141G, S024R-T274I, P086W-K235F, A015T-T033S, Y209A-K235F, S024R-R247H, S078R-K235F, S024R-V104A, T033S-A048T, G118R-K235F, T033S-T253A, T143A-Y209A, T033S-P239T, Y209A-N243F, S024R-A272P, and S024R-R269C, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
51 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: V004R-S009A-G020R-S242R, G020R-N043R-W241R, G020R-S242R-N269R, V004R-S009A-G020R-N043R, V004R-G020R-H249R, N018R-S024R-V244R, S009A-T022R-S212F-W241R, G020R-N043R-N269R, N018R-S024R-S242R, V004R-S009A-N043R-W241R, G020R-N043R-V244R, G020R-T022R-S242R, V004R-G020R-N043R, V004R-S009A-G020R-N043R-S242R, G020R-N043R-S242R, G020R-N043R-S242R-H249R, G020R-S212F-H249R, V004R-S009A-W241R, A001R-S009A-N043R, G020R-N043R-H249R, S009A-G020R-N043R-W241R, G020R-T022R-N043R, G020R-H249R-N269R, G020R-T022R-W241R, V004R-S009A-S024R-N043R-W241R, S009A-N043R-S078R, V004R-G020R-S024R-V244R, G020R-T022R-S078R-S242R, G020R-S024R-S242R-H249R, V004R-S009A-S078R-W241R, S009A-N043R-S078R-S242R, V004R-G020R-S024R, S009A-N043R-S212F, G020R-N043R-S212F, S024R-S078R-S212F, S009A-G020R-S024R-N043R, S009A-T022R-N043R-S078R, G020R-T022R-S212F-W241R, G020R-N043R-S212F-W241R, S009A-N043R-W241R, G020R-N043R-E271L, G020R-T022R-S078R-W241R, G020R-S024R-N043R-S242R, G020R-T022R-N043R-W241R, S009A-G020R-N043R-S212F, V004R-S009A-G020R-S024R-S242R, G020R-N043R-H249R-E271L, G020R-T022R-S024R-S242R, S009A-T022R-S078R-S212F, G020R-N043R-S242R-E271L, S009A-T022R-S078R-S212F-W241R, V004R-G020R-S024R-H249R, G020R-T022R-E271L, G020R-T022R-N043R-S212F, V004R-G020R-S024R-N043R-S242R, V004R-G020R-S024R-N043R, V004R-S009A-T022R-S078R-S212F, G020R-T022R-S078R-S212F-W241R, and G020R-T022R-N269R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
52 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: N018R-G020R-N043D-R045T-A230E, N018R-N043R-R045T-S242R-H249R, S024R-N043D-H249R, N018R-G020R-R045T, G020R-S024R-N076D-H249R, S024R-N043R-A230E-S242R, N018R-S024R-N043D-A230E, G020R-N076D, N018R-S024R-N043D-N076D-H249R, S024R-N043R-N076D-H249R, N018R-S024R-R045T-S242R, G020R-N043D-N076D-A230E-H249R, G020R-N043R-R045T-S242R, N018R-S024R-N076D-H249R, N018R-G020R-S024R-N043D-R045T-L233I-S242R, S024R-N043R-A230E, N018R-G020R-N043D, N043R-S242R-H249R, G020R-N043R-R045T-A230E, N043R-N076D-S242R-H249R, G020R-S024R-R045T-A230E-S242R, S024R-R045T-N076D-A230E-S242R-H249R, S024R-R045T, S024R-N043R-R045T-N076D-A230E-H249R, N018R-S024R-N043D-R045T-H249R, N018R-N043R-R045T-H249R, S024R-N043R-S242R, N018R-G020R-N043R-N076D-H249R, G020R-S024R-N043D-H249R, G020R-N043R-A230E-S242R, G020R-N043R-S242R, N018R-N043R-N076D-A230E, G020R-S024R-N043D-S242R, G020R-N043R-A230E, N018R-G020R-N043R-N076D-S242R-H249R, N043D-R045T-N076D-H249R, N018R-N043R-S242R-H249R, N018R-G020R-N043R-R045T-S242R, N018R-G020R-N043D-A230E-S242R, G020R-S024R-N043R-R045T-H249R, S024R-N043R-H249R, G020R-S024R-K27E-N043R-N076D-A230E, S024R-N043R-R045T-S242R, N018R-G020R-S024R-N043R-R045T-N076D-A230E, G020R-N043R-N076D-A230E-H249R, N018R-N043R-R045T-S242R, G020R-S242R-H249R, N018R-N043R-N076D-A230E-S242R-H249R, N018R-S024R-N076D, G020R-S024R-K27R-N043D-S242R-H249R, N018R-G020R-S024R-N043D-N076D-S242R, N018R-N043R-N076D-S242R-H249R, N018R-S024R-N043D-A230E-H249R, N018R-G020R-N043D-H249R, N018R-G020R-N043D-R045T-N076D-S242R, S024R-N043R-N076D-A230E-S242R, G020R-S024R-T381-N043R-R045T-N076D-S242R-H249R, N018R-G020R-N043R, N018R-S024R-R045T-A230E-S242R, N018R-G020R-H249R, S024R-N043R-N076D, N018R-G020R-S024R-N043R-R045T-N076D-H249R, N018R-N043D-R045T-N076D-S242R-H249R, S024R-N043D-S242R-H249R, N018R-G020R-S024R-N043D-R045T-S242R, G020R-S024R-N043R-N076D, N018R-G020R-N043D-R045T-A230E-S242R, G020R-S024R-N043R-R045T-N076D-S242R-H249R, N018R-N043R-R045T-N076D-S242R, N018R-G020R-N043R-N076D-A230E-S242R, N018R-S024R-N043D-H249R, N018R-S024R-N043R-R045T-A230E-H249R, N018R-G020R-N043R-R045T-N076D-H249R, N018R-S024R-S242R, N018R-N043R-R045T-N076D-A230E-S242R, R045T-S242R-H249R, N018R-S024R-N043D-S242R, N018R-G020R-N043D-R045T-S240P, S024R-N043R-R045T-S242R-H249R, N018R-S024R-V30S-L31S-D32I-T33Q-G34V-I35F, N018R-G020R-N043R-N076D, G020R-N043D-R045T-N076D-S242R-H249R, N018R-S024R-N043D-A230E-S242R, N018R-S024R-N043D-S242R-H249R, S024R-N043D-R045T-S242R-H249R, N043R-A230E-H249R, S024R-N043R-N076D-A230E-H249R, G020R-S024R-N043D-N076D-H249R, S024R-R045T-S242R-A273V, G020R-S024R-R045T-N076D-S242R-H249R, N018R-S024R-N043D-N076D-S242R, N018R-N043R-N076D-A230E-H249R, N018R-G020R-N043R-R045T-H249R, N018R-N043R-R045T-A230E-S242R, G020R-S024R-N043D-R045T-A230E-S242R, N018R-N043D-A230E-H249R, N018R-N043R-N076D-S242R, N018R-G020R-N076D, N018R-G020R-N043D-N076D-S242R-H249R, G020R-S024R-N043D-N076D-S242R-H249R, N043D-S242R-H249R, N018R-G020R-S024R-N043R-N076D, N018R-G020R-N043D-R045T-N076D-H249R, N018R-G020R-N043R-R045T-N076D-A230E-H249R, N018R-N076D-S242R, G020R-N043R-H249R, N018R-N076D-S242R-H249R, N018R-S024R-R045T-A230E-H249R, A230E-H249R, N018R-R045T-H249R, G020R-N043R-N076D, N043R-R045T-H249R, N018R-N043D-N076D-S242R-H249R, N043R-N076D-H249R, N018R-R045T, G020R-N076D-A230E-S242R, G020R-S024R-N043D-R045T, S024R-N043D-N076D-S242R-H249R, G020R-R045T-H249R, N043R-N076D-S153A-H249R, N043R-N076D-A230E-H249R, N018R-N043D-N076D-H249R, and G020R-N043R-N076D-V227I, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
53 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I-A232V-Q236H-Q245R-N252K, S101G-S103A-V104I-A232V-Q245R-N248R, S101G-S103A-V104I-G159R-A232V-Q245R-N248D, S101G-S103A-V104I-G159D-A232V-Q245R-N248R, S101G-S103A-V104I-A232V-Q245R, S101G-S103A-V104I-G159D-A232V-Q245R, and S101G-S103A-V104I-A232V-Q245R-N248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
54 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-P129E-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-L148I-A158E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-P129E-A232V-Q245R-N248D, S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-L148I-A158E-A232V-Q245R-N248D, A016S-S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A158E-G159E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-S128N-A158E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-A158E-S188D-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-P129E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-P129E-A158E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-P129E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-G159E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A158E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-L148I-A158E-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-A158E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S128N-P129E-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-P129E-G159E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-L148I-A158E-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-G159E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-G159E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-S128N-G159E-S188D-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-G159E-S188D-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-A158E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-G159E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-L148I-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-A158E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-A158E-G159E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-A158E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D, S101G-S103A-V104I-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-L148I-A158E-A232V-Q245R-N248D, S101G-S103A-V104I-P129E-A158E-G159E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-G159E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-A158E-G159E-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-A232V-Q245R-N248D, S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-L148I-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-G159E-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-S128N-P129E-A158E-A232V-N238R-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-S128N-P129E-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128N-A158E-G159E-S188D-A232V-Q245R-N248D, T022A-S024K-S101G-S103A-V104I-S128N-A158E-G159E-A232V-Q245R-N248D, S101G-S103A-V104I-P129E-L148I-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-L148I-A232V-Q245R-N248D, T022A-S101G-S103A-V104I-L148I-S188D-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-S128N-P129E-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-S128N-P129E-A158E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-L148I-A158E-S188D-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-L148I-A158E-S188D-A232V-Q245R-N248D, S101G-S103A-V104I-L148I-G159E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D, and S101G-S103A-V104I-S128N-P129E-A158E-A232V-Q245R-N248D-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
55 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: T022A-S024R-S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-G159E-S188D-A232V-Q245R-N248D-H249R, T022A-S024R-S101G-S103A-V104I-A158E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-G159E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-L148I-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-L148I-A158E-A232V-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D-H249R, A016S-S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129E-G159E-A232V-N238R-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-P129E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-L148I-A158E-A232V-Q245R-N248D, S024R-S101G-S103A-V104I-P129E-S188D-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-A158E-G159E-S188D-A232V-N238R-Q245R-N248D, T022A-S101G-S103A-V104I-A158E-G159E-A232V-N238R-Q245R-N248D, T022A-S024R-S101G-S103A-V104I-P129E-A158E-A232V-Q245R-N248D, S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, T022A-S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D-H249R, and S024R-S101G-S103A-V104I-A158E-G159E-A232V-Q245R-N248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
56 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: V104L-S128N-A158E-R186H-H249R, S128N-A158E-S188D-H249R, N062E-S128N-A158E-G159E-E271F, N062E-A158E-S188D-H249R-E271F, N062E-A158E-R186H-H249R-E271F, S128N-A158E-S188D-Y209E-E271F, N062E-G159E-S188D-H249R, A016S-N062E-A158E-R186H-H249R, N062E-A158E-G159E-H249R, S101A-S128N-A158E-Y209E-H249R, S128N-A158E-R186H-E271F, N062E-A158E-S188D-H249R, N062E-A158E-R186H-E271F, N062E-A158E-R186H-H249R, N062E-S101A-R186H-H249R, N062E-S101A-A158E-R186H-E271F, N062E-V104L-A158E-S188D-H249R-E271F, N062E-G159E-R186H-H249R, N062E-G159E-H249R, S128N-A158E-R186H-H249R, S128N-A158E-S188D-E271F, N062E-A158E-H249R, N062E-R186H-S188D-H249R-E271F, S128N-A158E-Y209E-, N062E-S101A-A158E-H249R, V104L-S128N-A158E-R186H-E271F, N062E-S101A-A158E-R186H-H249R-E271F, A016S-N062E-A158E-H249R, N062E-S101A-G159E-H249R, S128N-A158E-R186H-S188D-E271F, S101A-S128N-A158E-R186H-E271F, N062E-S101A-S188D-H249R, S101A-V104L-A158E-R186H-S188D-H249R, N062E-G159E-H249R-E271F, S128N-A158E-G159E-E271F, A016S-N062E-V104L-A158E-R186H-E271F, T022A-S128N-A158E-H249R, S128N-A158E-H249R, N062E-S101A-V104L-A158E-R186H-E271F, A016S-N062E-A158E-R186H-E271F, V104L-S128N-A158E-H249R, V104L-S128N-A158E-S188D-H249R, T022A-N062E-A158E, N062E-S101A-S188D-H249R-E271F, N062E-A158E-H249R-E271F, V104L-S128N-A158E-R186H-S188D-E271F, N062E-S101A-R186H-E271F, N062E-V104L-G159E-H249R, N062E-R186H-H249R, N062E-S101A-R186H-H249R-E271F, S101A-A158E-R186H-S188D-H249R, N062E-S101A-R186H, S101A-S128N-P129E-R186H-H249R, S101A-S103G-A158E-R186H-H249R, A016S-N062E-V104L-R186H-S188D-E271F, V104L-A158E-R186H-H249R, S101A-S128N-A158E-S188D-Y209E-E271F, N062E-S101A-R186H-S188D-E271F, A016S-N062E-A158E-H249R-E271F, N062E-S128N-A158E, N062E-S128N-G159E-H249R, N062E-S101A-A158E-S188D-H249R, S101A-S128N-A158E-H249R, N062E-A158E-R186H-S188D-H249R, A016S-V104L-A158E-R186H-E271F, N062E-L148I-G159E, N062E-S101A-A158E-R186H-H249R, N062E-S101A-R186H-S188D-H249R, V104L-A158E-R186H-S188D-H249R, N062E-S101A-V104L-R186H-S188D-E271F, T022A-S101A-A158E-R186H-H249R, S101A-S128N-A158E-Y209E, A158E-R186H-S188D-H249R-E271F, V104L-A158E-R186H-S188D-H249R-E271F, S101A-V104L-A158E-R186H-H249R, V104L-A158E-H249R, S101A-V104L-S128N-A158E-R186H-E271F, A016S-V104L-S188D-H249R, S101A-V104L-A158E-R186H-S188D-E271F, V104L-S128N-G159E-E271F, V104L-A158E-R186H-H249R-E271F, A158E-R186H-H249R, S101A-A158E-R186H-H249R, V104L-A158E-S188D-H249R-E271F, A016S-S128N-A158E-R186H, V104L-S128N-R186H-S188D-H249R, A016S-S101A-S128N-R186H, A016S-N062E-S128N-R186H-E271F, A016S-S128N-R186H-E271F, S128N-P129E-R186H, A158E-R186H-H249R-E271F, A016S-A158E-H249R, A016S-A158E-R186H-H249R, A016S-T022A-A158E-R186H-E271F, E089P-S101A-P129E-R186H, T022A-S128N-A158E-R186H, S101A-V104L-S128N-A158E-R186H, T022A-S128N-R186H-S188D-, N062E-V104L-A158E-R186H-S188D-H249R, T022A-A158E-R186H-H249R-E271F, T022A-V104L-A158E-H249R, S101A-L111V-P129E, A016S-A158E-H249R-E271F, A016S-L111V-S188D-, T022A-V104L-R186H-S188D-H249R, and V104L-L148I-S188D-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
57 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: A001R-S101G-S103A-V104I-A232V-Q245R, V004R-S101G-S103A-V104I-A232V-Q245R, N043R-S101G-S103A-V104I-A232V-Q245R-E271L, S078R-S101G-S103A-V104I-A232V-Q245R, V004R-N043R-S101G-S103A-V104I-A232V-Q245R, N018R-N043R-S101G-S103A-V104I-A232V-Q245R, G020R-S101G-S103A-V104I-A232V-Q245R, S101G-S103A-V104I-A232V-Q245R-E271L, G020R-N043R-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-S101G-S103A-V104I-A232V-Q245R, G020R-G025R-N116A-Y167W, N018R-S101G-S103A-V104I-A232V-Q245R, T022R-S101G-S103A-V104I-A232V-Q245R, S078R-S103N-S106G-Y167W-Q236N, N018R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-S101A-H120E-A194F-H249R, G020R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, S101G-S103A-V104I-S212F-A232V-Q245R, G020R-S144R-N185I-L233C-Q236N, G023A-S078R-S216F-Q236N-H249R, S101G-S103A-V104I-A232V-Q245R-N269R, S101G-S103A-V104I-G115R-A232V-Q245R, P052N-S078R-S103N-L148I-T213A, N018R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, G025R-E089I-N116A-P239S-A270C, S024R-S101G-S103A-V104I-A232V-Q245R, L148I-T213A-N252R, S024R-G025R-N183D-Y192W-P239S, G046R-A194F-S212M, V104L-L217E-T224A-H249R-N252R, G023A-Y091F-V121F-Y192W-Q236N, S101G-S103A-V104I-A232V-V244R-Q245R, S099F-S144R-Y167W-N252R, S101G-S103A-V104I-A232V-Q245R-H249R, N043R-S101G-S103A-V104I-A232V-Q245R, T022W-S078R-Y167W-S212M-A270C, V121F-N252R-A270C, G020R-S103N-S216F-Q236N-N252R, N043R-S101G-S103A-V104I-A232V-Q245R-H249R, G023A-P052N-Y192W-I198L-N252R, G025R-G046R-V121F, S024R-S078R-V104L-N116A-N183D, G046R-Q059A-S103N-G211Q-S212M, G020R-P052N-N062Q-Y091F-Y192W, G023A-P052N-S144R-Y192W-S216F, S101G-S103A-V104I-A232V-S242R-Q245R, P052N-S103N-N116A-L148I-Y192W, E089I-N116A-N117F-T224A-H249R, S144R-G211Q-N238L-P239S-H249R, N043A-N062Q-A194F-G211Q, G020R-S024R-P052N-Q059A-S216F, S024R-Y167W-T224A-H249R, T057R-Y167W-H249R, G025R-S103N-R186K-A194F-T224A, S105T-S128N-S144R-L148I-S212M, G020R-Q059A-S144R-Y192W-T224A, S024R-N043A-N117F-A194F-G211Q, N117F-A194F-T213A-A270C, S078R-Y091F-V121F-L233C-N252R, T057R-S099F-S105T-I198L-T213A, G023A-Y091F-S101A-I198L-N252R, N062Q-S103N-V121F-S144R-H249R, N043R-S101G-S103A-V104I-A232V-S242R-Q245R, G023A-S024R-N117F-S212M-S216F, V104L-T213A-S216F, A194F-G211Q-Q236N, N062Q-S103N-N117F-A194F, S024R-N062Q-V104L-S106G-H249R, T057R-E089I-I198L, G046R-Q059A-S106G-L217E-H249R, N117F-T213A-A215F, S101A-H120E-Y192W-A215F-T224A, N043A-T057R-N117F-S144R-N183D, G046R-N183D-N238L, G025R-N043A-E089I-N117F, S078R-V104L-T213A-A215F-T224A, Y091F-S099F-S101A-S105T-Y167W, S106G-N117F-N238L, G046R-E089I-Y091F-S101A-N116A, G020R-N062Q-E089I-R186K-S212M, T057R-S099F-V121F-N185I-Y192W, G046R-E089I-Y192W-L233C-A270C, E089I-N117F-N185I-A215F-L233C, P052N-V104L-N183D-S216F-H249R, S078R-S099F-N116A-R186K-T224A, G025R-S105T-S128N-S144R-A270C, S105T-G211Q-S216F, S024R-G046R-Y091F-V121F, S106G-N185I-S216F-Q236N, N062Q-S101A-Q236N-N252R-A270C, G025R-N043A-Y091F-I198L-A270C, G020R-G023A-V104L-Y192W-L233C, S024R-N043A-S105T-S106G-I198L, G020R-E089I-L217E, S024R-Y091F-I198L-A215F-P239S, G046R-E089I-S099F-R186K-S212M, V104L-H120E-R186K-S216F-N252R, T022W-A194F-T213A-L233C-N238L, S099F-S105T-S106G-A194F-S212M, E089I-S105T-N116A-A215F-S216F, G025R-N116A-H120E-T224A-A270C, N043A-Q059A-S101A-S216F-T224A, T057R-N183D-Q236N, G025R-N062Q-S128N-S144R-N185I, S103N-H120E-Y167W-I198L-L233C, T022W-E089I-S216F, S024R-S106G-N116A-S212M-T224A, G020R-P052N-S101A-I198L-L233C, E089I-Y091F-N185I-G211Q-A270C, L111I-A215F-P239S, S024R-N116A-R186K-L233C-Q236N, and G023A-S103N-S106G-S212M-A215F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
58 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-T022W-S078R-S101A-S103A-V104I-N116S-T213A-A215F-A232V-Q245R, N018R-S078R-S101G-S103A-V104I-A232V-Q245R, S024R-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-A232V-Q245R, G020R-T22W-S101G-S103A-V104I-A232V-Q245R, N018R-N043R-S101G-S103A-V104I-A232V-Q245R, N018R-T022W-S024R-N076D-S101A-N116A-A232V-Q245R, N018R-V104I-A232V-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-H249R, N018R-N043D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-N043R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-N043D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S103A-A232V-H249R, N018R-S101G-V104I-A232V-Q245R, G020R-S024R-S101G-S103A-V104I-L217E-A232V-Q245R-H249R, N018R-T22K-N043D-S101G-S103A-V104I-A232V-Q245R, N043R-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-T22W-S101G-S103A-V104I-G211Q-A232V-Q245R, S024R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-T22W-S078R-S101A-S103A-V104I-N116A-N183D-A232V, N018R-S024R-N076D-N116A-A215F-H249R, N018R-N043R-R045T-S101G-S103A-V104I-A232V-Q245R, S024R-N043R-N076D-S101G-S103A-V104I-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-A232V-Q245R, G020R-T022W-S101G-S103A-V104I-G211Q-A232V-Q245R, G020R-T022W-S078R-S101G-S103A-V104I-N116A-T213A-A215F-A232V-Q245R, N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S024R-N076D-S101A-N116A-T213A-H249R, N018R-S024R-N076D-N116A-G211Q-H249R, N043R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-S101G-Q245R, G020R-T22W-S101A-S103A-V104I-G211Q-T213A-A232V-Q245R, G020R-S024R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-S078R-S101G-S103A-V104I-G211Q-T213A-A215F-A232V-Q245R, R045T-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-S101G-S103A-H249R, N018R-T22W-S024R-N076D-S101A-N116A-A232V-Q245R, N018R-S101G-V104I-A232V-H249R, G020R-T22W-S101A-S103A-V104I-A215F-A232V-Q245R, N018R-S024R-N076D-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-S101A-I198L-H249R, S024R-S101G-S103A-V104I-A232V-Q245R, G020R-N076D-S101G-S103A-V104I-A232V-Q245R-N269R, G020R-S101G-S103A-V104I-A232V-Q245R-N269R, N043D-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-S101G-V104I-T213A-A215F-A232V-Q245R, G020R-S101G-S103A-V104I-N116A-A215F-A232V-Q245R, S024R-S103A-V104I-H249R, N018R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-S101G-V104I-Q245R, G020R-S101G-S103A-V104I-G211Q-T213A-A215F-A232V-Q245R, S024R-S103A-V104I-A232V-H249R, N018R-S024R-N076D-N116A-G211Q-A215F-H249R, N018R-Q245R, S024R-S103A-Q245R, S024R-S103A-V104I-Q245R, G020R-S078R-S101G-A232V-Q245R, N018R-S024R-N076D-V104I-H249R, N018R-S024R-V104I-H249R, S024R-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N076D-G211Q-A215F-H249R, R019H-G020R-T022W-S078R-S101G-S103A-V104I-G211Q-A232V-Q245R, N018R-S024R-N076D-S101A-I198L-G211Q-T213A-H249R, N018R-S024R-N043D-S101G-S103A-V104I-A232V-Q245R, G020R-T22W-S103A-V104I-A232V-Q245R, N018R-S103A-V104I-H249R, N018R-T022W-S024R-N076D-S101A-I198L-A215F-H249R, N018R-S024R-S101G-V104I-A232V, S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S024R-N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-N043D-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-T22W-S024R-N076D-N116A-T213A-H249R, N018R-S024R-S101G-V104I, G020R-S101A-S103A-V104I-A215F-A232V-Q245R, N018R-R045T-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-S101G-S103A-Q245R, N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-G211Q-A215F-A232V-Q245R, 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N018R-G020R-S024R-N076D-S101A-N116A-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N183D-I198L-H249R, N018R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-S024R-N076D-N116A-N183D-T213A-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-H249R, N018R-S024R-N076D-S101G-Q245R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-T213A-A215F-H249R, N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-N116A-I198L-T213A-A215F-H249R, N076D-V104I-A232V-Q245R, K027R-N043R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-T022W-S024R-N076D-S101A-N183D-T213A-A215F-H249R, N018R-S024R-N076D-S103A-V104I-L135I-A232V, N018R-G020R-T022W-S024R-N076D-N183D-I198L-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-G211Q-H249R, N018R-T022W-S024R-N076D-N183D-G211Q-T213A-A215F-H249R, N076D-S101G-S103A-V104I-A232V-H249R, P500S-N018R-T022W-S024R-N076D-S101A-T213A-A215F-H249R, N018R-S024R-N076D-N116A-N183D-A215F-H249R, N018R-S024R-N076D-N183D-I198L-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-H249R, N018R-T022W-S024R-N076D-N116A-N183D-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N183D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-N116A-I198L-H249R, N018R-S024R-N076D-N183D-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-N183D-T213A-H249R, N018R-S024R-N076D-N116A-N183D-I198L-A215F-H249R, N018R-T022W-S024R-N076D-N183D-I198L-G211Q-A215F-H249R, N018R-T022W-S024R-N076D-N183D-H249R, N018R-T022W-S024R-N076D-S101A-N183D-I198L-A215F-H249R, N018R-G020R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, G020R-T022W-S101A-S103A-V104I-N183D-I198L-A215F-A232V-Q245R, N076D-S101G-S103A-V104I-H249R, N018R-G020R-S024R-N076D-N183D-I198L-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-A215F-H249R, N018R-G020R-S024R-N076D-G211Q-T213A-H249R, N018R-S024R-N076D-N116A-N183D-G211Q-T213A-H249R, N018R-S024R-N076D-N116A-N183D-I198L-H249R, N018R-G020R-S024R-N076D-G211Q-N243D-H249R, N018R-G020R-R045T-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-T022W-S024R-N076D-N183D-I198L-G211Q-T213A-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-N116A-I198L-G211Q-T213A-A215F-H249R, S024R-N076D-S101G, N018R-G020R-S024R-N076D-S101A-I198L-T213A-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-N183D-A215F-H249R, N018R-N043D-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-T022W-S024R-N076D-N116A-N183D-G211Q-T213A-Q245R, G020R-S024R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-R045T-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-S024R-N076D-S101A-N183D-I198L-G211Q-H249R, N018R-T022W-S024R-N076D-N183D-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-N116A-I198L-G211Q-A215F-H249R-N269S, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-A215F-H249R, G020R-T022W-S101A-S103A-V104I-N183D-T213A-A215F-A232V-Q245R, N018R-S024R-N076D-A086V-S101A-N183D-I198L-G211Q-H249R, N018R-N076D-S101G-I198T-A232V, N018R-G020R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N183D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-N183D-H249R-A248T, N018R-N076D-V104I-Q245R-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-S101A-H249R, N018R-G020R-S024R-N076D-S101A-N116A-T213A-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-G211Q-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-T213A-H249R, N043R-R045T-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N043R-R045T-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-N076D-S101G-V104I, G020R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-H249R, N043D-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-H249R, N018R-N076D-V104I, N018R-G020R-N043D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-T022W-S024R-N076D-N183D-G211Q-H249R, N018R-S024R-N076D-S101A-N183D-I198L-G211Q-A215F-H249R, S101G-V104I, N018R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-S101A-N183D-G211Q-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N116A-N183D-A215F-H249R-N263D, N018R-T022W-S024R-N076D-S101A-N183D-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-A215F-H249R, N018R-T022W-S024R-N076D-N116A-I198L-A215F-H249R-R269H, N018R-N043D-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-R045T-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-I198L-T213A-A215F-H249R, N018R-G020R-S024R-N076D-N183D-I198L-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-G211Q-H249R, N018R-G020R-T022W-S024R-N076D-S101A-I198L-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-H249R, G020R-T022W-S101G-S103A-V104I-N116A-N183D-I198L-G211Q-T213A-A215F-A232V-Q245R, V104I-A232V, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-N183D-I198L-G211Q-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N183D-I198L-H249R, N043D-R045T-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-G211Q-H249R, N018R-G020R-N043R-S101G-S103A-V104I-L217E-A232V-Q245R, S024R-N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-R045T-N076D-S078R-S101G-S103A-V104I-Y166F-A176P-A179V-N184T-A187P-A194P, N018R-T022W-S024R-D041E-N076D-S101A-S160T-N183D-G211Q-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-T213A-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-I198L-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N183D-G211Q-A215F-H249R, S024R-N076D-V104I, N018R-N076D-S101G-A232V, N018R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-A215F-H249R, N018R-S024R-L031F-N076D-N116A-N183D-G211Q-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-T213A-H249R-N269S, N018R-G020R-S024R-N076D-S101A-N116A-I198L-A215F-H249R, N018R-N043R-N076D-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N076D-S101A-N114T-I198L-G211Q-A215F-H249R, S024R-N043R-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, S024R-N076D-S103A-V104I-A232V, N018R-G020R-T022W-S024R-N076D-S101A-I198L-G211Q-A215F-H249R, S101G-V104I-A232V, N018R-T022W-S024R-N076D-N183D-I198L-T213A-A215F-H249R, N018R-T022W-S024R-N076D-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N183D-T213A-H249R, N018R-S024R-N076D-S101A-N183D-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-G211Q-T213A-H249R, N018R-S078R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-G211Q-A215F-H249R-T260A, N076D-H249R, N018R-T022W-S024R-N076D-N183D-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N116A-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-T213A-H249R, N018R-G020R-T022W-S024R-N076D-N116A-N183D-I198L-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N183D-H249R, N076D-V104I-H249R, G020R-T022W-S101A-S103A-V104I-N116A-N183D-G211Q-T213A-A215F-A232V-Q245R, S024R-N043R-R045T-S078R-S101G-S103A-V104I-L217E-A232V-Q245R, N018R-G020R-T022W-S024R-N076D-N183D-I198L-A215F-H249R, N018R-N076D-S101G-S103A, G020R-S024R-S101G-S103A-V104I-A232V-Q245R-N269R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-G211Q-H249R, S101G-S103A-A232V, S024R-N076D-S101G-A232V, N018R-T022W-S024R-N076D-S101A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-G211Q-A215F-H249R, N018R-G020R-S024R-N076D-N183D-I198L-H249R, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-H249R, N018R-G020R-S024R-N076D-N116A-A156V-N183D-G211Q-A215F-H249R-N269S, R045T-N076D-S101G-S103A-V104I-A232V-Q245R-H249R, N018R-G020R-S024R-N076D-S101A-N183D-I198L-H249R, N018R-G020R-T022W-S024R-N076D-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-G211Q-A215F-H249R, N018R-G020R-T022W-S024R-N076D-N116A-H249R, N018R-N076D-S101G, N018R-S024R-N076D-N116A-N183D-I198L-T213A-A215F-H249R, N018R-G020R-T022W-S024R-N076D-S101A-N116A-I198L-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-H249R, N018R-N076D-A232V, N018R-G020R-S024R-N076D-S101A-A232V-Q245R, N018R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, N018R-T022W-S024R-N076D-N183D-T213A-A215F-H249R, N018R-S024R-N076D-S101A-N116A-G211Q-T213A-N237D-H249R, N018R-G020R-S024R-N076D-S101A-N116A-N183D-G211Q-H249R-R275S, N018R-T022W-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-A215F-H249R, S024R-N076D, N018R-S024R-N076D-N183D-G211Q-A215F-H249R, N018R-T022W-S024R-N076D-N116A-N183D-I198L-T213A-A215F-H249R, N076D-V104I-A232V-H249R, N018R-N076D-S103A-A232V, N018R-G020R-S024R-N076D-S101A-N116A-N183D-I198L-G211Q-T213A-H249R, N018R-G020R-S024R-N076D-S101A-N183D-T213A-H249R, N018R-G020R-S024R-N076D-S101A-D175E-N183D-G211Q-A215F-H249R, N018R-G020R-N043D-S078R-S101G-S103A-V104I-L217E-A232V-Q245R-A273E, G020R-S024R-N043D-R045T-N076D-S101G-S103A-V104I-A232V-Q245R, P005S-S101G-S103A-V104I-A232V-Q245R-H249R, S103A-V104I-A232V, N018R-G020R-S024R-V068A-N076D-S101A-N116A-T213A-A215F-H249R, N018R-T022W-S024R-N076D-S101A-I198L-A215F-H249R-R275S, N018R-S024R-N076D-N183D-I198L-G211Q-T213A-H249R, and N043D-R045T-S101G-S103A-V104I-A232V-Q245R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
59 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: N018R-S024R-N043R-N076D-H249R-N269R, N018R-T022R-S024R-N043R-N076D-H249R, N018R-N043D-S101G-S103A-V104I-A232V-Q245R, G020R-N043D-S101G-S103A-V104I-A232V-Q245R, N043D-S101G-S103A-V104I-A232V-Q245R-N269R, N043D-S078R-S101G-S103A-V104I-A232V-Q245R, N043R-N076D-S101G-S103A-V104I-A232V-Q245R, T022R-N043R-S101G-S103A-V104I-A232V-Q245R, N043R-S078R-S101G-S103A-V104I-A232V-Q245R, G020R-N076D-S101G-S103A-V104I-A232V-Q245R, N043R-N076D-S101G-S103A-V104I-A232V-Q245R, T022R-N076D-S101G-S103A-V104I-A232V-Q245R, N076D-S078R-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N043R-N076D-H249R, N018R-S024R-N076D-S242R-H249R, N018R-S024R-N076D-H249R-N269R, N018R-T022R-S024R-N076D-H249R, N018R-S024R-N076D-S078R-H249R, N018R-S024R-N043D-N076D-H249R-N269R, N018R-T022R-S024R-N043D-N076D-H249R, N018R-S024R-N043D-N076D-S078R-H249R, G020R-S101G-S103G-V104I-A232V-Q245R, G020R-S101G-S103A-V104L-A232V-Q245R, G020R-S101G-S103A-V104V-A232V-Q245R, G020R-S101G-S103S-V104I-A232V-Q245R, G020R-S101G-S103S-V104L-A232V-Q245R, G020R-S101S-S103S-V104I-A232V-Q245R, G020R-S101S-S103S-V104L-A232V-Q245R, G020R-S101A-S103A-V104L-A232V-Q245R, G020R-S101S-S103S-V104V-A232V-Q245R, G020R-S101S-S103A-V104I-A232V-Q245R, G020R-S101S-S103A-V104V-A232V-Q245R, G020R-S101S-S103G-V104I-A232V-Q245R, G020R-S101S-S103G-V104V-A232V-Q245R, G020R-S101A-S103A-V104V-A232V-Q245R, G020R-S101A-S103S-V104I-A232V-Q245R, G020R-S101A-S103S-V104V-A232V-Q245R, N018R-S024R-N043R-N076D-S078R-H249R, S024R-N043D-S101G-S103A-V104I-A232V-Q245R, N043D-S101G-S103A-V104I-A232V-Q245R-H249R, S024R-N076D-S101G-S103A-V104I-A232V-Q245R, N076D-S101G-S103A-V104I-A232V-S242R-Q245R, N018R-G020R-S024R-N076D-L217E-H249R, N018R-S024R-N043R-N076D-L217E-H249R, N018R-S024R-N043D-N076D-S242R-H249R, N018R-G020R-S024R-N043R-N076D-H249R, G020R-S101A-S103G-V104V-A232V-Q245R, N043D-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N076D-L217E-H249R-N269R, and N018R-S024R-N076D-L217E-S242R-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
60 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-S101A-S103A-V104I-G118R-A232V-Q245R, G020R-S024R-N116A-T213A, N043R-S101A-N116A-A215F-N269R, S024R-N043R-S101A-N116A, S024R-N043R-S101A-N116A-A215F-N269R, G020R-S101G-S103A-V104I-A215F-A232V-Q245R, N043R-S101A-N269R, S024R-N043R-N116A-T213A-N269R, G020R-S024R-N043R-R045T-S101A-T213A, S024R-N043R-N116A-A215F-N269R, G020R-S024R-T213A-A215F, G020R-N116A-N269R, S024R-N116A-T213A-N269R, N043R-S101A-N116A-N269R, S101G-S103A-V104I-N116A-T213A-A232V-Q245R-N269R, S024R-N043R-R045T-S101A-N116A-A215F-N269R, G020R-N043R-S101A-N269R, S101A-S103A-V104I-T213A-A232V-Q245R-N269R, S024R-A215F-N269R, N043R-S101A-N116A-T213A-A215F-N269R, N043R-S101A-T213A-N269R, G020R-S024R-N043R-R045T-N116A-T213A, S101G-S103A-V104I-A232V-Q245R-N269R, S024R-N043R-R045T-S101A-N116A-T213A-N269R, S024R-N043R-R045T-N269R, G020R-N043R-R045T-S101A-N269R, S024R-N043R-N116A-N269R, G020R-S024R-N043R-R045T, N043R-N116A-N269R, S024R-N043R-S101A-A215F-N269R, S024R-N043R-R045T-T213A-A215F-N269R, G020R-S024R-R045T-N269R, G020R-N043R-S101A-N116A-T213A-A215F, G020R-S101G-S103A-V104I-T213A-A215F-A232V-Q245R, G020R-S024R-R045T-N116A-N269R, G020R-S101A-N116A-N269R, S024R-N043R-A215F, G020R-S024R-T213A, S024R-N043R-S101A-A215F, G020R-S024R-N043R-R045T-N116A, G020R-S024R-N043R-R045T-S101A-N269R, G020R-S024R-S101A-A215F, G020R-S024R-N116A-T213A-A215F, G020R-S024R-N116A, G020R-S024R-S101A-N116A, N043R-T213A-A215F-N269R, S024R-S101A-N269R, S024R-N043R-N116A-A215F, G020R-T038A-N043R-S101A, G020R-S024R-N116A-A215F, S024R-N043R-S101A-T213A, P014L-G020R-S024R-N043R-R045T-S101A-A215F, G020R-S024R-A215F, G020R-N116A-A215F-N269R, G020R-R045T-N116A-N269R, G020R-S024R-N043R-R045T-A215F, and G020R-S024R-N043R-R045T-N116A-T213A-A215F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
61 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: N043R-N076D-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, S024R-N043R-N076D-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F-E271F, S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R-E271F, N076D-S101G-S103A-V104I-A114V-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, S024R-N076D-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, S024R-N043R-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-N043R-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101A-S103A-V104I-A158E-S166D-S188D-L217E-A232V-Q245R-N248D-H249R, N076D-S101G-S103A-V104I-S128L-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, and N043R-N076D-S101G-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
62 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: T022A-S101G-S103A-V104I-G159D-L217E-A232V-Q245R-N248D-E271F, T022A-N043R-S101G-S103A-V104I-G159D-S188D-L217E-A232V-Q245R-N248D-E271F, T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, N043R-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, N043R-N076D-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R-E271F, S024R-S101G-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-N043R-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, T022A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, N076D-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, N043R-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R, S024R-N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, N076D-S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, N043R-N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S101G-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R-E271F, S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, S101G-S103A-V104I-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-A158E-S188D-A232V-Q245R-N248D-H249R, N076D-S101G-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R-E271F, N043R-N076D-S101A-S103A-V104I-A158E-S166D-S188D-A232V-Q245R-N248D-H249R, and N076D-S101A-S103A-V104I-A158E-S188D-L217E-A232V-Q245R-N248D-H249R-E271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
63 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: H017R-T022A-N076D-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-N043R-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, T022A-S101G-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-H249R-E271F, H017R-T022A-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, T022A-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R-E271F, T022A-S101G-G102A-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, and T022A-N043R-N076D-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R-E271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
64 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101S-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103G-V104V-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103S-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103A-V104L-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103G-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103G-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103S-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101S-S103S-V104V-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103S-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103S-V104I-G159E-A232V-Q245R-N248D-H249R, S101S-S103A-V104I-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104L-A158E-S188D-A232V-Q245R-N248D-H249R, S101A-S103A-V104L-G159E-A232V-Q245R-N248D-H249R, S101A-S103S-V104L-G159E-A232V-Q245R-N248D-H249R, S101G-S103S-V104L-G159E-A232V-Q245R-N248D-H249R, S101S-S103A-V104L-G159E-A232V-Q245R-N248D-H249R, S101A-S103G-V104V-G159E-A232V-Q245R-N248D-H249R, and S101S-S103A-V104V-G159E-A232V-Q245R-N248D-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
65 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: V026F-V051W-V104L-S106E, V026F-L031F-S078N-G102A-S160D, G020K-G100S-N116L-A158E-S166D-N243F, T033S-N043W-N218D-P239G-N243F, T022L-T038F-A048R-N062E-G100S-R186K, S101D-S103N-N116L-S144R-A215D, V104L-S105T-T213A-L217E-S256N, N043W-S101D-S212M-N243F, V026F-A048R-S105T-T213A-N218D-T224A, S024F-S101D-G118R-A215D-L250I-A272F, V121F-N185E-T224A-P239G, T022L-L031F-G102A-S128D-T224A-N243F, N062E-S078N-G102A-N116L-S144R-L250I, T022L-T038F-V121F-S160D-A272F, V026F-S078N-G159C-R186K-N243F, S024F-A048R-G118R-S166D-L217E, G023A-T038F-S078N-G100S-S212M-A215D, G100S-N116L-A158E-T213A, S078N-V104L-G118R-S128D, G102A-S103N-S105T-A194E, T022L-S078N-S128D-T213A, K027R-G100S-G118R-S160D-S188D-N243F, S024F-G102A-R186K-T213A-L217E-N243F, T033S-S105T-S188D-S216F, G023A-G100S-A194E-S212M, A048R-S128D-N185E-P239G, G020K-S024F-T033S-P129E-A194E, G020K-K027R-P129E-S166D-P239G, T022L-G023A-K027R-S101D-V104L-S216F, T033S-G118R-P129E-A194E-P239G, T022L-S078N-N116L-P129E-S256N, K027R-S101D-S103N-S105T-A272F, A048R-S078N-N116L-N185E-L217E-P239G, G023A-S024F-K027R-N062E, S024F-S103N-V104L-G118R-S188D, V026F-V104L-S256N-A272F, S024F-N043W-V104L-V121F-P129E, N062E-S078N-N116L-T224A, G023A-S024F-V051W-A158E, K027R-T038F-G102A-N116L, N062E-S078N-S144R-S212M, L031F-N116L-S256N-A272F, T022L-T033S-V104L-N116L-S160D-R186K, S024F-G118R-P129E-R186K-T213A, N043W-S105T-T213A-A215D-S216F, L031F-S105T-R186K-S188D, V026F-A194E-T213A-S256N, and S103N-S160D-L250I-S256N, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
66 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: T022A-S024R-S101D-S103A-V104I-G118R-G159D-S188D-A232V-N248D-E271F, T022A-S024R-S103A-V104I-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S024R-S103A-V104I-G118R-G159D-S188D-L217D-A232V-N248D, T022A-S024R-S101D-S103A-V104I-G118R-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-S101D-S103A-V104I-G159D-S188D-A232V-Q245R-N248D, T022A-N043R-S103A-V104I-G118R-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-N043R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-A232V-N248D, T022A-N043R-S101D-S103A-V104I-G118R-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S024R-N043R-S101D-S103A-V104I-G159D-S188D-A232V-Q245R-N248D, T022A-S103A-V104I-G159D-S188D-A232V-N248D, T022A-S024R-S103A-V104I-G118R-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S024R-S103A-V104I-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, T022A-N043R-N062E-S103A-V104I-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-N043R-S103A-V104I-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-S103A-V104I-G159D-S188D-L217D-A232V-N248D-E271F, T022A-S103A-V104I-G118R-G159D-S188D-L217D-A232V-Q245R-N248D, T022A-S024R-S101D-S103A-V104I-G118R-S128I-G159D-S188D-A232V-Q245R-N248D, T022A-S024R-N043R-S103A-V104I-G159D-S188D-L217D-A232V-N248D-E271F, T022A-N043R-S103A-V104I-G118R-G159D-S188D-L217D-A232V-N248D-E271F, T022A-N043R-S103A-V104I-G118R-G159D-S188D-A232V-N248D-E271F, T022A-S103A-V104I-S128I-P129E-G159D-S188D-A232V-N248D-E271F, T022A-S103A-V104I-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, T022A-N043R-S103A-V104I-S128I-G159D-S188D-A232V-Q245R-N248D, T022A-S101D-S103A-V104I-G118R-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, T022A-S103A-V104I-G118R-P129E-G159D-S188D-A232V-Q245R-N248D-E271F, T022A-S024R-N043R-S103A-V104I-G118R-G159D-S188D-L217D-A232V-N248D, T022A-N062E-S103A-V104I-G118R-G159D-S188D-A232V-Q245R-N248D, T022A-N043R-S101D-S103A-V104I-G118R-P129E-G159D-S188D-L217D-A232V, T022A-S024R-S103A-V104I-G159D-S188D-L217D-A232V-N248D, T022A-S024R-N043R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-L217D-A232V-N248D-E271F, and T022A-S103A-V104I-G118R-G159D-S188D-L217D-A232V-Q245R-N248D-E271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
67 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020K-S024F-N062E-S188D-P239G, S024F-N062E-N116L-P239G, G020K-G023A-N062E-S188D, G020K-G023A-S024F-N062E-G118R-S188D-T213A, G020K-N043W-N062E-N116L-S188D-T213A-P239G, G023A-N062E-N116L-G118R, G023A-S024F-N062E-N116L-G118R, S024F-N116L, S024F-N062E-S188D-T213A, G023A-N062E-N116L-G118R-S188D-P239G, G020K-S024F-N062E, G020K-N043W-N062E-N116L-P239G, S024F-N062E-N116L-T213A-P239G, G020K-S024F-N043W-N062E-N116L-T213A, G020K-G023A-S024F-N062E-N116L-S188D-T213A, S024F-N062E-S188D-P239G, G023A-N043W-N062E-N116L-G118R-T213A, N062E-S188D-P239G, G020K-S024F-N062E-P239G, S024F-N116L-G118R-S188D-P239G, G020K-G023A-N062E-N116L-G118R-T213A, G020K-G023A-S024F-N062E-S188D-T213A-P239G, S024F-N043W-G118R-S188D, G023A-S024F-N116L-G118R-S188D-T213A, G020K-G023A-N043W-N116L-S188D-T213A-P239G, G023A-S024F-N116L-S188D-P239G, G023A-N043W-N116L-G118R-S188D, G023A-S024F-G118R-S188D-P239G, G023A-S024F-N043W-N062E-N116L-G118R, G020K-N043W-S188D-T213A, S024F-N062E-G118R-P239G, G023A-N043W-S188D-T213A, G020K-S024F-N043W-N062E-N116L-G118R-S188D-P239G, G020K-N116L-S188D-P239G, G020K-N043W-N062E-G118R, G020K-N043W-N116L-S188D-T213A, G020K-S024F, G023A-N043W-N116L-P239G, G023A-S024F-N043W-N116L-G118R-S188D-P239G, G020K-G023A-N043W-T213A, and G023A-S024F-N062E-G118R-T213A-P239G, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
68 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020K-G023A-N043W-G118R-S128I-P129E-G159D-S188D, S024F-G118R-S128I-P129E-G159D, G020K-S024F-N062E-N116L-G118R-S188D, G020K-N062E-N116L-S188D, N062E-N116L-G118R-T213A, G020K-G023A-N062E-N116L-S188D, N062E-N116L-G118R-S188D, G020K-N062E-N116L-T213A, G020K-G023A-N062E-N116L, G020K-N062E-S188D-T213A, G020K-N062E, G020K-S024F-N062E-N116L-S188D, G020K-N043W-N062E-N116L-S188D, G020K-S024F-N062E-S188D-T213A, N062E-N116L-S188D-T213A, G020K-N062E-N116L, G020K-G023A-N062E-N116L-S188D-T213A, G023A-S024F-N062E-N116L-T213A, T022A-N043R-S103A-V104I-S128I-P129E-G159D-S188D-A232V-Q245R-N248D, T022A-N043R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-A232V-N248D-E271F, S024F-N062E-N116L-S188D, T022A-S024R-S103A-V104I-G118R-S128I-P129E-G159D-S188D-A232V-N248D, G023A-N062E-N116L-S188D, N043W-N062E-N116L, G020K-G023A-N116L-S188D, N043W-N062E-N116L-S188D, S024F-N062E-N116L, N062E-N116L-S188D, and T022A-S024R-S103A-V104I-S128I-G159D-S188D-A232V-N248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
69 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S087R-S101G-S103A-V104I-Q109R-S212P-A232V-Q245R-E271V, S101G-S103A-V104I-Q109R-A232V-Q245R, S101G-S103A-V104I-Q109R-S212P-A232V-Q245R-E271V, S101G-S103A-V104I-Q109R-S212P-A232V-Q245R, N076D-S87R-S103A-V104I-S212P-E271V, N076D-S103A-V104I-Q109R, N076D-S103A-V104I-S212P-E271V, N076D-S103A-V104I-Q109R-Q245R, and N076D-S103A-V104I-S212P-Q245L-E271V, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO: 1.
70 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S024R-P086W-G118R, S024R-S078R-P086W-N243F, S024R-T033S-P086S-S087N-Y209A, T033S-G118R, S024R-S078R-P086W-G118R-A270T, S024R-T033S-P086W-G118R, S078R-P086W-N243F, T033S-S078R-P086W-G118R-Y209A, T033S-S078R-Y209A, P086W-G118R-N243F, S024R-P086W, S078R-P086W-K235F, S024R-G118R, S024R-P086R, S101G-S103A-V104I-A232V, S024R-T033S-S078R-P086W-G118R, S024R-G118R-Y209A, Y209A-W241R, T033S-P086W-N243F, T033S-A172V-Y209A, G118R-Y209A-N243F, S024R-P086S-S141G, S024R-G118R-Y209A-N243F, S024R-T033S-P086S-S085N-K235F, S024R-T033S-A133V, S024R-T033S-S078R-P086W, S024R-P086W-Y209A, S024R-W241R, T033S-G118R-N243F, S024R-K235F, S024R-S078R-P086W, S024R-G118R-Y209A-K235F, S024R-Y209A-W241R, T033S-G118R-W241R, P086W-G118R-Y209A, T033S-G118R-G159D-Y209A, T033S-S078R-P086W, S024R-P086W-N243F, G118R-Y209A, S024R-P086W-G118R-V203I, S078R-Y209A-K235F, S024R-T033S-W241R, S078R-G118R, T033S-G118R-Y209A-N243F, L021M-S024R-T033S, S024R-T033S-P086W, T033S-K235F, S078R-P086W-Y209A, S024R-T033S-Y209A-K235F, T033S-P086W-G118R, S024R-T033S-S078R-Y209A, T033S-P086W-G118R-Y209A-N243F, P086W-Y209A-N243F, P005S-S078R-G118R-W241R, S024R-A174T, T033S-Y209A-N243F, P086W-G118R-A133V, S024R-T033S-G118R, S024R-P086W-Y209A-K235F, P086W-Y209A, I008T-S024R, P086W-G118R, T033S-W241R, P005S-S024R-T033S-N243F, S024R-Y209A-S242P, S024R-T033S-S078R-G118R, S024R-T033S-A194T, S024R-N243F, S024R-Y209A, S024R-T033S-G118R-Y209A, T033S-P086W, S024R-T033S, S024R-T033S-S078R-N243F, P086W-N243F, T033S-G118D-A138V-Y209A, T033S-Y209A-K235F, S024R-P086R-G118R, T033S-P201S, S024R-P239Q, T033S-G118R-Y209A-, S078R-P086W, K235F-N243F, S024R-Y209A-K235F, G118R-A172V, H017Y-S024R-T033S-P086W, T033S-L148F, S024R-G118R-K235F, T033S-S078R, T033S-N243F, S024C-T033S, G118R-A194T, T033S-Y209A, G118R-Y209A-K235F, S024R-T033S-Y209A-N243F, S024R-T033S-K235F, S024R-T033S-G118R-K235F, S024R-S141G, S024R-T274I, S024R-T033S-Y209A, P086W-K235F, S024R-Y209A-N243F, V004E-T033S-S078R, P086W-Y209A-K235F, A015T-T033S, T033S-P086W-S156L-Y209A, S024R-G118R-N243F-R269H, Y209A-K235F, S024R-R247H, S024R-T033S-A228T, S078R-K235F, S024R-T033S-A174V-K235F, S024R-K235F-N243F, S024R-T033S-K235F-W241R, S024R-T033S-A151V, S024R-V104A, T033S-A048T, Q012H-V104A-G118R, G118R-K235F, T033S-T253A, T143A-Y209A, S024R-T033S-N243F, T033S-P239T, Y209A-N243F, S024R-T033S-P129H-N184D-T253M, S024R-A085V-P086W-G118R-K235F, S024R-A272P, and S024R-R269C, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
71 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: G020R-S087D-S101G-S103A-V104I-A232V-Q245R, G020R-S101G-S103A-V104I-V150L-A232V-Q245R, N018R-G020R-S024R-N076D-S087D-H249R, N018R-G020R-S024R-N076D-V150L-H249R, N018R-S024R-N043R-N076D-S087D-H249R, N018R-S024R-N043R-N076D-V150L-H249R, N018R-S024R-N076D-S078R-S087D-H249R, N018R-S024R-N076D-S078R-V150L-H249R, N018R-S024R-N076D-S087D-H249R-N269R, N018R-S024R-N076D-S087D-S242R-H249R, N018R-S024R-N076D-S087D-V150L-H249R, N018R-S024R-N076D-V150L-H249R, N018R-S087D-S101G-S103A-V104I-A232V-Q245R, N018R-S101G-S103A-V104I-V150L-A232V-Q245R, N018R-T022R-S024R-N076D-S087D-H249R, N018R-T022R-S024R-N076D-V150L-H249R, N043R-S087D-S101G-S103A-V104I-A232V-Q245R-N269R, N043R-S101G-S103A-V104I-V150L-A232V-Q245R, S024R-S087D-S101G-S103A-V104I-A232V-Q245R, S024R-S101G-S103A-V104I-V150L-A232V-Q245R, S078R-S087D-S101G-S103A-V104I-A232V-Q245R, S078R-S101G-S103A-V104I-V150L-A232V-Q245R, S087D-S101G-S103A-V104I-A232V-Q245R-N269R, S101G-S103A-V104I-V150L-A232V-Q245R-H249R, S101G-S103A-V104I-V150L-A232V-Q245R-N269R, T022R-S087D-S101G-S103A-V104I-A232V-Q245R, N018R-S024R-N043D-N076D-V150L-H249R, N043R-S087D-S101G-S103A-V104I-A232V-Q245R, T022R-S101G-S103A-V104I-V150L-A232V-Q245R, N018R-S024R-N043D-N076D-S087D-H249R, N018R-S024R-N076D-S087D-H249R, N018R-S024R-N076D-V150L-S242R-H249R, N043R-S101G-S103A-V104I-V150L-A232V-Q245R-N269R, N076D-S101G-S103A-V104I-V150L-A232V-Q245R, S087D-S101G-S103A-V104I-A232V-S242R-Q245R, S101G-S103A-V104I-V150L-A232V-Q245R, N076D-S087D-S101G-S103A-V104I-A232V-Q245R, S087D-S101G-S103A-V104I-A232V-Q245R, and S101G-S103A-V104I-V150L-A232V-S242R-Q245R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
72 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S024R-S101G-S103A-V104I-P129Q-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S130A-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-S130A-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, and S024R-K027R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
73 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I-A232V-M222Q-Q245R, S101G-S103A-V104I-A158E-S188D-M222S-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-S188D-M222Q-A232V-Q245R-N248D-H249R, N076D-S101G-S103A-V104I-A232V-M222Q-Q245R, S101G-S103A-V104I-A232V-M222S-Q245R, N076D-S101G-S103A-V104I-A232V-M222S-Q245R, and N076D-S101G-S103A-V104I-A158E-S188D-M222S-A232V-Q245R-N248D-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
74 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S024R-S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S130A-A158E-N183D-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-P129Q-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-S128L-S130A-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-S130A-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-P129Q-A158E-S188D-A232V-Q245R-N248D-H249R-E271G, S101G-S103A-V104I-P129Q-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S130A-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, S024R-S101G-S103A-V104I-S128L-A158E-N183D-S188D-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-P129Q-A158E-S188D-L217E-A232V-Q245R-N248D-H249R, and S024R-S101G-S103A-V104I-S128L-S130A-A158E-S188D-A232V-Q245R-N248D-H249R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
75 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I, T22A-S101A-Y209E, S103G-L111V-G159E, T22A-S103G-G159E, T22A-L111V-G159E, T22A-S128N-E271F-Y209E, T22A-S103G-L111V, N62E-L111V-S128N, T22A-L111V-S128N, T22A-N62E-L111V, S101A-S103G-V104L-S188D, S101G-S103A-V104I-G159D, S101A-S103G-V104L-S128N, T22A-S101A-G159E, S101A-S103G-V104L, S101A-S103G-V104L-G159E, T22A-S101A-S103G-V104L, S101A-S103G-V104L-Y209E, T22A-Y209E-E271F, T22A-S101A-E271F, and S101A-Y209E-E271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
76 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S103A-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K, S101G-V104I-G159D-A232V-Q236H-Q245R-N248D-N252K, S101G-S103A-G159D-A232V-Q236H-Q245R-N248D-N252K, S101G-S103A-V104L-A232V-Q236H-Q245R-N248D-N252K, S101G-S103A-V104L-G159D-Q236H-Q245R-N248D-N252K, S101G-S103A-V104L-G159D-A232V-Q245R-N248D-N252K, S101G-S103A-V104L-G159D-A232V-Q236H-N248D-N252K, S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N252K, and S101G-S103A-V104L-G159D-A232V-Q236H-Q245R-N248D, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
77 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I-G159D-A232V-Q245R-N248D, S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N238R, S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N248R, S101G-S103A-V104I-G159D-A232V-Q245R-N248D-T253R, S101G-S103A-V104I-G159D-A232V-Q245R-N248D-S24R, S101G-S103A-V104I-G159D-A232V-Q245R-N248D-N76D, S101G-S103A-V104I-G159E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-G159E-A232V-Q245R-N248D-E271F, S101G-S103A-V104I-A158E-A232V-Q245R-N248D-H249R, S101G-S103A-V104I-A158E-A232V-Q245R-N248D-E271F, T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R, T22A-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-H249R, and N62E-S101G-S103A-V104I-G159D-A232V-Q245R-N248D-E271F, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
78 . An isolated subtilisin variant having proteolytic activity comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: A1R, A230E, E271L, G115R, G20R, H249R, K235F, K27V/F/L, L75E, L82R, N18R, N269R, N43D, N43R, N76D, R45T, S212F, S242R, S24R, S78R, S9A, T22R, V121E, V244R, V28E, V30E, V4R, W241R, G20R-N43R-H249R, G20R-T22R-N43R, G20R-N43R-S242R, G20R-N43R-E271L, G20R-N43R-V244R, G20R-S24R-N43R-S242R, S9A-T22R-S78R-S212F-W241R, S9A-G20R-N43R-S212F, S9A-N43R-S212F, G20R-N43R-S212F, G20R-T22R-N43R-S212F, S24R-S78R-S212F, S9A-N43R-S78R, S9A-N43R-S78R-S242R, S9A-G20R-N43R-S78R, G20R-S24R-N43R-S78R-S242R, T22R-S24R-S78R-S212F, S9A-G20R-N43R-S78R-S242R, G20R-N43R-S78R-H249R, G20R-N43R-S78R, S9A-S78R-S212F, S9A-T22R-N43R-S78R, S9A-G20R-S24R-N43R, S9A-T22R-S78R-S212F, V4R-S9A-T22R-S78R-S212F, G20R-S24R-N43R, A1R-S9A-N43R, G20R-S24R-N43R-G115R, S9A-S24R-N43R, G20R-T22R-S24R-N43R, A1R-S24R-N43R, S9A-G20R-S24R-N43R-S242R, S9A-G20R-T22R-S78R-S212F, S9A-S24R-N43R-V244R, S9A-S24R-N43R-S242R, V4R-S9A-T22R-S24R-S212F, and T22R-S24R-N43R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
79 . An isolated subtilisin variant having proteolytic activity and comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: S101G-S103A-V104I-A232V-Q245R-N248D, S101G-S103A-V104I-G159D-A232V-Q245R, S101G-S103A-V104I-G159R-A232V-Q245R-N248D, S101G-S103A-V104I-G159D-A232V-Q245R-N248R, S101G-S103A-V104I-A232V-Q245R, S101G-S103A-V104I-A232V-Q245R-N248R, S101G-S103A-V104I-G159R-A232V-Q245R-N248R, S101G, S103A, V104I, A232V, Q236H, and Q245R, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
80 . An isolated subtilisin variant having proteolytic activity and comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: A16S, T22A, S24R, N62E, N76D, E89P, S101A/G, S103G/A, V104L/I, L111V, S128N, P129E, A232V, L148I, A158E, G159D/E, R186H, S188D, Y209E, Q236H, Q245R, N248D/R, H249R, N252K/R, T253R, and E271F wherein amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
81 . An isolated subtilisin variant having proteolytic activity and comprising an amino acid sequence comprising a combination of amino acid substitutions selected from: A1R, Q2S, Q2M, Q2A, Q2R, Q2W, S3R, V4R, V4S, V4C, I8A, S9A, S9F, S9W, R10S, R10A, R10H, R10M, Q12F, Q12R, P14K, P14F, P14Q, A15R, A15F, A16S, H17R, H17M, H17F, N18R, N18K, G20F, G20K, G20R, T22A, T22R, T22Y, T22V, T22Q, T22L, T22W, G23A, G23S, G23F, S24R, S24F, S24W, S24Q, S24H, S24L, G25V, G25F, G25R, V26F, K27L, K27F, K27R, K27V, V28A, V28N, V28E, A29T, V30E, L31F, T33S, T33G, T33D, G34P, I35M, S36T, S36F, S36R, T38L, T38F, T38R, P40N, P40L, P40T, P40W, P40H, P40R, L42I, N43A, N43F, N43I, N43S, N43R, N43M, N43W, N43D, R45T, G46R, A48R, F50C, V51W, V51F, V51H, P52F, P52E, P52N, P55Y, T57R, Q59A, Q59F, Q59R, D60P, D60Q, D60A, N62E, N62Q, G63V, G63M, G63T, G63I, G63A, G63S, G63H, G63Q, G63D, G63E, G63P, H64F, H64T, V68A, V68C, A69N, A69T, A69P, A69W, T71G, T71G, I72C, A74C, L75A, L75F, L75E, L75R, N76D, S78R, S78N, S78I, S78R, I79W, I79Q, V81R, L82F, L82T, L82V, L82R, L82M, A85M, P86W, P86L, P86I, E89P, E89T, E89G, E89H, E89W, E89L, E89V, E89W, E89F, E89I, Y91N, Y91F, A92F, K94N, S99F, S99T, S99P, S99G, S99M, G100S, G100N, G100Q, G100I, S101A, S101N, S101G, S101D, S101T, S101D, S101E, S101P, S101F, G102A, G102T, G102N, G102H, G102E, S103G, S103N, S103D, S103A, V104L, V104I, V104E, V104D, S105T, S105E, S105Q, S106G, S106T, S106E, S106D, S106A, S106V, S106F, I107M, I107F, A108I, A108G, Q109M, L111V, L111I, E112V, E112L, E112Q, A114G, G115K, G115R, N116K, N116A, N116L, N117F, G118R, G118I, M119C, H120A, H120F, H120R, V121F, V121F, V121E, N123G, N123E, L124S, S128D, S128F, S128L, S128N, S128H, S128M, S128I, S128Q, P129E, S132A, S132E, A138G, S144R, V147L, L148I, A158E, G159D, G159E, G159C, S160D, S166D, S166E, Y167W, M175V, V177C, D181A, Q182R, N183I, N183D, N183M, N183R, N183F, N183R, N185E, N185V, N185I, R186H, R186K, S188E, S188D, S188R, Y192H, Y192W, A194E, A194V, A194F, D197F, D197F, I198L, I198F, V203E, V203C, T208S, Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G, Y209L, P210R, P210V, P210L, G211Q, G211R, S212I, S212M, S212F, T213A, Y214F, A215N, A215D, A215E, A215H, A215F, S216F, S216A, L217E, L217N, L217D, N218D, N218P, N218E, T224A, T224G, V227I, A230E, A231I, A231C, A232V, L233C, V234F, K235F, Q236F, Q236N, Q236H, N238R, N238K, N238L, P239K, P239G, P239R, P239R, P239H, P239T, P239N, P239S, P239F, S240R, W241R, S242L, S242R, N243F, N243R, V244R, Q245R, I246S, N248D, N248V, N248I, N248R, H249R, H249T, L250I, K251R, K251S, N252I, N252F, N252R, N252K, N252H, T253I, T253R, T253F, A254C, S256N, G258R, T260V, T260I, L262D, L262H, Y263F, S265F, L267V, L267N, L267M, N269I, N269R, A270C, E271I, E271H, E271V, E271H, E271M, E271L, E271P, E271A, E271F, E271T, A272F, A272F, A272R, A273F, A273I, and T274G, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1.
82 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, and wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations at amino acid positions selected from amino acid 1, 2, 3, 4, 8, 9, 10, 12, 14, 15, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, 36, 38, 40, 42, 43, 45, 46, 48, 50, 51, 52, 55, 57, 59, 60, 62, 63, 64, 68, 69, 71, 72, 74, 75, 76, 78, 79, 81, 82, 85, 86, 89, 91, 92, 94, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 128, 129, 132, 138, 44, 147, 148, 158, 159, 160, 166, 167, 175, 177, 181, 182, 183, 185, 186, 188, 192, 194, 197, 198, 203, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 224, 227, 230, 231, 233, 234, 235, 236, 236, 238, 238, 239, 240, 241, 242, 243, 244, 246, 248, 249, 250, 251, 252, 253, 254, 256, 258, 260, 262, 263, 265, 267, 269, 270, 271, 272, 273, and 274, wherein the amino acid positions of the subtilisin variant are numbered by correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' set forth as SEQ ID NO:1, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the variant protease amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
83 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, and wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations selected from the group of A1R, Q2S, Q2M, Q2A, Q2R, Q2W, S3R, V4R, V4S, V4C, I8A, S9A, S9F, S9W, R10S, R10A, R10H, R10M, Q12F, Q12R, P14K, P14F, P14Q, A15R, A15F, A16S, H17R, H17M, H17F, N18R, N18K, G20F, G20K, G20R, T22A, T22R, T22Y, T22V, T22Q, T22L, T22W, G23A, G23S, G23F, S24R, S24F, S24W, S24Q, S24H, S24L, G25V, G25F, G25R, V26F, K27L, K27F, K27R, K27V, V28A, V28N, V28E, A29T, V30E, L31F, T33S, T33G, T33D, G34P, I35M, S36T, S36F, S36R, T38L, T38F, T38R, P40N, P40L, P40T, P40W, P40H, P40R, L42I, N43A, N43F, N43I, N43S, N43R, N43M, N43W, N43D, R45T, G46R, A48R, F50C, V51W, V51F, V51H, P52F, P52E, P52N, P55Y, T57R, Q59A, Q59F, Q59R, D60P, D60Q, D60A, N62E, N62Q, G63V, G63M, G63T, G63I, G63A, G63S, G63H, G63Q, G63D, G63E, G63P, H64F, H64T, V68A, V68C, A69N, A69T, A69P, A69W, T71G, T71G, I72C, A74C, L75A, L75F, L75E, L75R, N76D, S78R, S78N, S78I, S78R, I79W, I79Q, V81R, L82F, L82T, L82V, L82R, L82M, A85M, P86W, P86L, P86I, E89P, E89T, E89G, E89H, E89W, E89L, E89V, E89W, E89F, E89I, Y91N, Y91F, A92F, K94N, S99F, S99T, S99P, S99G, S99M, G100S, G100N, G100Q, G100I, S101A, S101N, S101G, S101D, S101T, S101D, S101E, S101P, S101F, G102A, G102T, G102N, G102H, G102E, S103G, S103N, S103D, S103A, V104L, V104I, V104E, V104D, S105T, S105E, S105Q, S106G, S106T, S106E, S106D, S106A, S106V, S106F, I107M, I107F, A108I, A108G, Q109M, L111V, L111I, E112V, E112L, E112Q, A114G, G115K, G115R, N116K, N116A, N116L, N117F, G118R, G118I, M119C, H120A, H120F, H120R, V121F, V121F, V121E, N123G, N123E, L124S, S128D, S128F, S128L, S128N, S128H, S128M, S128I, S128Q, P129E, S132A, S132E, A138G, S144R, V147L, L148I, A158E, G159D, G159E, G159C, S160D, S166D, S166E, Y167W, M175V, V177C, D181A, Q182R, N183I, N183D, N183M, N183R, N183F, N183R, N185E, N185V, N185I, R186H, R186K, S188E, S188D, S188R, Y192H, Y192W, A194E, A194V, A194F, D197F, D197F, I198L, I198F, V203E, V203C, T208S, Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G, Y209L, P210R, P210V, P210L, G211Q, G211R, S212I, S212M, S212F, T213A, Y214F, A215N, A215D, A215E, A215H, A215F, S216F, S216A, L217E, L217N, L217D, N218D, N218P, N218E, T224A, T224G, V227I, A230E, A231I, A231C, A232V, L233C, V234F, K235F, Q236F, Q236N, Q236H, N238R, N238K, N238L, P239K, P239G, P239R, P239R, P239H, P239T, P239N, P239S, P239F, S240R, W241R, S242L, S242R, N243F, N243R, V244R, Q245R, I246S, N248D, N248V, N248I, N248R, H249R, H249T, L250I, K251R, K251S, N252I, N252F, N252R, N252K, N252H, T253I, T253R, T253F, A254C, S256N, G258R, T260V, T260I, L262D, L262H, Y263F, S265F, L267V, L267N, L267M, N269I, N269R, A270C, E271I, E271H, E271V, E271H, E271M, E271L, E271P, E271A, E271F, E271T, A272F, A272F, A272R, A273F, A273I, and T274G, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
84 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations selected from the group of A1R, Q2S, V4R, V4S, S9A, R10S, P14K, A16S, H17R, N18R, G20R, T22A, T22R, S24R, S24W, G25R, G25V, V26F, L42I, N43R, N43A, G46R, P52F, P52E, P52N, T57R, Q59A, N62E, N62Q, V68A, V68C, T71G, I72C, A74C. L75A, L75F, L75R, N76D, S78R, L82R, P86W, E89P, E89T, E89G, E89H, E89I, E89V, E89W, Y91N, K94N, G100S, S101A, S101N, S101G, S101D, S103G, S103N, V104L, V104I, S106V, S106G, A108I, L111V, E112V, G115K, G115R, N117F, G118I, V121F, S128D, S128F, S128L, S128N, P129E, S144R, L148I, A158E, G159E, S160D, S166D, N185E, N185I, R186H, S188E, S188D, D197F, V203E, Y209S, Y209N, Y209F, Y209T, Y209E, Y209H, Y209G, P210R, S212I, S212F, Y214F, A215N, A215D, A215E, L217E, L217N, T224A, A230E, A231I, Q236F, N238R, N238K, P239K, P239G, P239R, P239S, W241R, S242R, S242L, N243R, V244R, N248I, N248V, H249R, L250I, N252R, T253R, L262D, Y263F, S265F, L267V, L267N. N269I, N269R, E271F, E271I, E271H, E271P, E271T, E271V, E271L and A272F, and optionally comprises at least one mutation selected from the group of S103A, G159D, Q236H, Q245R, N248D and N252K, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
85 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations selected from the group of G20K, G20R, G23A, S24F, S24R, N43R, N43W, R45T, N62E, N76D, S101A, N116A, N116L, G118R, S128I, P129E, S188D, T213A, A215F, L217E, P239G, and N269R, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
86 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, and wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations selected from the group of A16S, T22A, S24R, N62E, N76D, E89P, S101A/G, S103G/A, V104L/I, L111V, S128N, P129E, A232V, L148I, A158E, G159D/E, R186H, S188D, Y209E, Q236H, Q245R, N248D/R, H249R, N252K/R, T253R, and E271F, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
87 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, and wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations selected from the group of A1R, A230E, E271L, G115R, G20R, H249R, K235F, K27V/F/L, L75E, L82R, N18R, N269R, N43D, N43R, N76D, R45T, S212F, S242R, S24R, S78R, S9A, T22R, V121E, V244R, V28E, V30E, V4R, and W241R, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
88 . A protease variant of Bacillus lentus subtilisin GG36 protease, wherein said Bacillus lentus subtilisin GG36 protease comprises the amino acid sequence shown in SEQ ID NO:2, and wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2 in no more than two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mutations selected from the group of A1R, A230E, E271L, G115R, G20R, H249R, K235F, K27V/F/L, L75E, L82R, N18R, N269R, N43D, N43R, N76D, R45T, S212F, S242R, S24R, S78R, S9A, T22R, V121E, V244R, V28E, V30E, V4R, and W241R, and optionally comprises at least one mutation selected from the group of S103A, G159D, Q236H, Q245R, N248D and N252K, wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
89 . The protease variant of any of claims 1 - 26 , wherein said protease variant comprises an amino acid sequence which differs from the amino acid sequence of SEQ ID NO:2, and wherein the total net charge of the protease variant is 0, +1, +2, +3, +4, +5, −1, −2, −3, −4, or −5 relative to the total net charge of the Bacillus lentus subtilisin GG36 protease, wherein the parent protease is GG36, and wherein amino acid positions of the protease variant are numbered according to the numbering of corresponding amino acid positions in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:1 as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
90 . The protease variant of any of claims 1 - 89 , wherein the protease variant has one or more of the following characteristics: a) a Test Method 2 performance index of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from 1.1 to about 5; b) a Test Method 3 performance index of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.1 to about 10, from 1.1 to about 8, or even from 1.1 to about 5; c) a Test Method 4 performance index of at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5; and/or d) a Test Method 6 performance index of at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from 1.0 to about 10, from 1.0 to about 8, or even from 1.0 to about 5.
91 . An isolated nucleic acid comprising a polynucleotide sequence that encodes the subtilisin variant provided in any of claims 1 - 90 .
92 . An expression vector comprising the nucleic acid of claim 91 .
93 . A host cell comprising the expression vector of claim 92 .
94 . A method for producing at least one subtilisin variant of a Bacillus subtilisin, comprising:
a) transforming a host cell with an expression vector comprising at least one nucleic acid encoding at least one subtilisin variant of any of claims 1 - 90 to produce a transformed host cell; and b) cultivating the transformed host cell under conditions suitable for the production of at least one subtilisin variant, to produce at least one subtilisin variant.
95 . The method of claim 94 , further comprising harvesting the produced subtilisin variant.
96 . The expression vector of claim 92 , wherein said at least one nucleic acid is operably linked to a promoter.
97 . The method of any of claims 94 - 95 , wherein said host cell is a Bacillus species.
98 . The method of claim 97 , wherein said Bacillus species is B. subtilis.
99 . A composition comprising at least one subtilisin variant of any of claims 1 - 90 , wherein said composition is not a fabric and home care product.
100 . The composition of claim 99 , wherein said composition is a cleaning composition.
101 . The composition of any of claims 99 - 100 , wherein said cleaning composition is a granular, powder, solid, bar, liquid, tablet, gel, or paste composition.
102 . The composition of any of claims 99 - 101 , further comprising at least one bleaching agent.
103 . The composition of any of claims 99 - 102 , wherein said cleaning composition is phosphate-free.
104 . The composition of any of claims 99 - 103 , wherein said cleaning composition contains phosphate.
105 . The composition of any of claims 99 - 104 , further comprising at least one additional enzyme.
106 . The composition of claim 105 , wherein said at least one additional enzyme is selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, and amylases, or any combination thereof.
107 . The composition of any of claims 99 - 106 , wherein said subtilisin variant is not a cold water protease variant.
108 . A method of cleaning, comprising contacting a surface or an item with a cleaning composition comprising at least one subtilisin variant of any of claims 1 - 90 .
109 . A method of cleaning comprising contacting a surface or an item with a cleaning composition set forth in any of claims 100 - 107 .
110 . The method of any of claims 108 - 109 , further comprising rinsing said surface or item after contacting said surface or item, respectively, with said cleaning composition.
111 . The method of any of claims 108 - 110 , further comprising the step of rinsing said surface or item after contacting said surface or item with said cleaning composition.
112 . The method of claim 111 , further comprising the step of drying said surface or item after said rinsing of said surface or item.
113 . A method of cleaning a surface or item, comprising: providing the cleaning composition set forth in any of claims 100 - 107 and a surface or item in need of cleaning; and contacting said cleaning composition with said surface or item in need of cleaning under conditions suitable for the cleansing of said surface of said surface or item, to produce a cleansed surface or item.
114 . The method of claim 113 , further comprising the step of rinsing said cleansed surface or item to produce a rinsed surface or item.
115 . The method of any of claims 113 - 114 , further comprising the step of drying said rinsed surface or item.
116 . The method of any of claims 108 - 115 , wherein said subtilisin variant is not a cold water protease variant.Cited by (0)
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