US2019194577A1PendingUtilityA1

Compositions and methods comprising serine protease variants

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Assignee: DANISCO US INC GENENCOR DIVPriority: Nov 11, 2008Filed: Sep 7, 2018Published: Jun 27, 2019
Est. expiryNov 11, 2028(~2.3 yrs left)· nominal 20-yr term from priority
Inventors:Luis G. Cascao-PereiraDavid A. EstellFrits GoedegebuurJames T. Kellis, Jr.Ayrookaran J. PouloseBrian F. SchmidtWolfgang Aehle
C12N 9/54C11D 3/38645C11D 3/386C12N 15/74C11D 3/38618C11D 3/38654C11D 3/38636C12Y 304/21062C11D 3/38609C12N 9/6424C12N 9/50
68
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Claims

Abstract

The present invention provides methods for protein engineering and 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-modified
1 - 18 . (canceled) 
     
     
         19 . An isolated subtilisin variant of a  Bacillus  subtilisin, wherein said subtilisin variant is a mature form having proteolytic activity and comprising substitutions selected from: S87N/Q109Q/G118D/S128L/P129Q/S130A/S188S/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213R/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213T/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213T/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213T/N248R, S87R/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248R, S87R/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213T/N248R, S87R/Q109R/G118R/S128L/P129Q/S130A/S188D/T213T/N248R, S87N/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213R/N248R, S87R/Q109Q/G118R/S128L/P129Q/S130A/S188S/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213T/N248N, S87R/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213E/N248R, S87D/Q109D/G118D/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213R/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213T/N248R, S87R/Q109R/G118R/S128L/P129Q/S130A/S188D/T213E/N248R, S87R/Q109Q/G118R/S128L/P129Q/S130A/S188D/T213E/N248N, S87N/Q109R/G118R/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213E/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213E/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213T/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213T/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213T/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213T/N248D, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213R/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213E/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213T/N248D, S87R/Q109D/G118D/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213R/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213R/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248N, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213E/N248N, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248N, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213R/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213T/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213R/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213T/N248N, S87R/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109D/G118V/S128L/P129Q/S130A/S188R/T213R/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213T/N248D, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213E/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213R/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213E/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188D/T213E/N248D, S87R/Q109D/G118R/S128L/P129Q/S130A/S188D/T213E/N248N, S87N/Q109R/G118V/S128L/P129Q/S130A/S188R/T213R/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213R/N248N, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213T/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213E/N248R, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213R/N248D, and S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213E/N248R, wherein the positions are numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         20 . The isolated subtilisin variant of  claim 19 , wherein said variant comprises a combination of substitutions selected from: S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213T/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213R/N248N, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213R/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213E/N248D, S87R/Q109D/G118R/S128L/P129Q/S130A/S188S/T213E/N248R, S87R/Q109D/G118R/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109D/G118V/S128L/P129Q/S130A/S188D/T213E/N248D, and S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213E/N248D,
 wherein the positions are numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1.   
     
     
         21 . The isolated subtilisin variant of  claim 19 , wherein said variant comprises a combination of substitutions selected from: S87D/Q109D/G118R/S128L/P129Q/S130A/5188R/T213E/N248D, S87N/Q109D/G118R/S128L/P129Q/S130A/S188D/T213E/N248R, S87D/Q109Q/G118D/S128L/P129Q/S130A/S188S/T213E/N248D, S87N/Q109R/G118V/S128L/P129Q/S130A/S188S/T213E/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188D/T213E/N248D, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188R/T213T/N248D, S87R/Q109D/G118V/S128L/P129Q/S130A/S188D/T213E/N248R, S87N/Q109Q/G118V/S128L/P129Q/S130A/S188S/T213R/N248D, and S87R/Q109D/G118R/S128L/P129Q/S130A/S188D/T213T/N248R, wherein the positions 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 of a  Bacillus  subtilisin, wherein said subtilisin variant is a mature form having proteolytic activity and comprising substitutions selected from: A001C/S024V/I107L, A001I/R045T/I107R/A172T, A001K/A172L, A001K/A172W, A001K/I072C/L126F, A001K/I072C/L126F/S164Q, A001K/I072C/V104I/L126F, A001K/I072C/V104I/L126F/S164Q, A001K/I072V, A001K/I072V/L126F, A001K/I072V/L126F/S164F, A001K/I072V/V104I, A001K/I072V/V104I/L126F/S164I, A001K/I072V/V104I/S164F, A001K/I072V/V104I/S164Q, A001K/I107T/A172Q, A001K/I107V/G127Q, A001K/I107V/G127Q/A172Q, A001K/I107V/G127R/A172Q, A001K/L126F, A001K/L126F/S164F, A001K/L126F/S164I, A001K/L126F/S164Q, A001K/R045F/I072C, A001K/R045F/I072C/L126F, A001K/R045F/I072C/V104I/S164F, A001K/R045F/I072V/L126F/S164I, A001K/R045F/I072V/L126F/S164Q, A001K/R045F/I072V/S164F, A001K/R045F/I072V/V104G/S164T, A001K/R045F/I107L/A172N, A001K/R045F/I107T, A001K/R045F/I107V/G127Q, A001K/R045F/L126F/S164F, A001K/R045F/L126F/S164I, A001K/R045F/S164F, A001K/R045F/V104I/L126F/S164Q, A001K/R045K/I072C/L126F/S164Q, A001K/R045K/I072V/L126F, A001K/R045K/I072V/L126F/S164F, A001K/R045K/I072V/L126F/S164Q, A001K/R045K/I072V/S164F, A001K/R045K/I072V/S164Q, A001K/R045K/I072V/V104I/L126F/S164F, A001K/R045K/I072V/V104I/L126F/S164I, A001K/R045K/I072V/V104I/L126F/S164Q, A001K/R045K/I107A/A172T, A001K/R045K/I107R/G127A/A172N, A001K/R045K/I107T/G127R, A001K/R045K/I107T/G127R/A172Q, A001K/R045K/L126F, A001K/R045K/L126F/S164I, A001K/R045K/L126F/S164P, A001K/R045K/L126F/S164Q, A001K/R045K/S164F, A001K/R045K/V104I/S164Q, A001K/R045K/V104L/S164A, A001K/R045N/I107T/G127Q, A001K/R045S/V104C/L126Y/S164F, A001K/S024H/I107T/G127Q, A001K/S024H/I107T/G127R, A001K/S024H/I107V, A001K/S024H/R045N/I107T, A001K/S024H/R045N/I107T/G127Q/A172Q, A001K/S024L/R045A, A001K/S024N/I107V/G127T, A001K/S024N/R045K/A172Y, A001K/S024R/I107T, A001K/S024R/R045N/A172C, A001K/S024R/R045N/I107T, A001K/S024R/R045N/I107T/G127R, A001K/S024T/I107T/G127Q/A172Q, A001K/S024T/R045N/G127Q/A172Q, A001K/S024W/I107T/G127R, A001K/S024W/I107V/G127T/A172Q, A001K/S164Q, A001K/V104E, A001K/V104I/L126F, A001K/V104I/L126F/S164I, A001K/V104I/L126F/S164Q, A001L/I072C, A001L/R045Q/G127H/A172R, A001L/S024D/R045Y/I107Y, A001M/R045S/I107E/A172D, A001P/A172V, A001Q/R045K/I107T, A001R/A172G, A001R/A172Q, A001R/I107T, A001R/I107T/A172Q, A001R/I107T/G127Q/A172Q, A001R/I107V/A172Q, A001R/R045F, A001R/R045K/A172Q, A001R/R045K/G127R, A001R/R045K/I107T/G127Q, A001R/R045K/I107V/G127R, A001R/R045N/A172K, A001R/R045N/A172Q, A001R/R045N/I107T, A001R/R045N/I107T/G127Q, A001R/R045N/I107V/A172V, A001R/R045N/I107V/G127Q/A172Q, A001R/R045N/I107V/G127R, A001R/S024H/I107T, A001R/S024H/R045K/I107T, A001R/S024H/R045K/I107T/A172Q, A001R/S024H/R045N/I107T/A172Q, A001R/S024N, A001R/S024R, A001R/S024R/A172H, A001R/S024R/A172Q, A001R/S024R/I107V/A172L, A001R/S024R/I107V/G127R, A001R/S024R/R045F/I107T/G127Q, A001R/S024R/R045F/I107T/G127R, A001R/S024R/R045K/I107T/G127Q, A001R/S024T/I107T/A172Q, A001R/S024T/R045K/A172Q, A001R/S024T/R045K/I107L/G127P/A172P, A001R/S024T/R045N/I107T/A172Q, A001R/S024W/I107V/A172S, A001R/S024W/R045F/I107V/G127Q, A001R/S024W/R045F/I107V/G127R, A001R/S024W/R045K, A001R/S024W/R045K/I107T/A172Q, A001R/S024W/R045N/I107T, A001S/A172V, A001S/S024L/I107Y/A172S, A001T/S024H/I107L/A172Y, A001V/I107L/A172I, G097P/N185Q/A215I, G097P/Y209W/A215V/S256W, G118L/G127R/S132L, G118T/G127Q, I107A/G127K/A172G, I107C/A172S, I107P/A172S, I107Q/A172D, L126A/S164N, L126F/S164V, M119F/N185R, M119F/N185R/Y209W/S256T, M119F/N185V/Y209W, M119F/S144T/N185V/S256I, M119F/S144W/N185R/S256L, M119F/Y209W, M119F/Y209W/S256I, N018K/A215I, N018K/A215R, N018K/A215V, N018K/G097P/A215I/S256W, N018K/G097P/N185K/S256W, N018K/G097P/N185K/Y209W/A215I/S256W, N018K/G097P/N185Q/Y209W/A215V/S256W, N018K/G097P/N185R/A215R/S256W, N018K/G097P/N185R/A215V/S256W, N018K/G097P/N185R/S256W, N018K/G097P/N185R/Y209W, N018K/G097P/N185R/Y209W/S256W, N018K/G097P/Y209W/A215V/S256W, N018K/N185K, N018K/N185K/A215V/S256W, N018K/N185K/S256W, N018K/N185K/Y209W/A215R, N018K/N185Q/A215R/S256W, N018K/N185Q/A215V/S256W, N018K/N185Q/S256W, N018K/N185Q/Y209W/S256W, N018K/N185R/A215I/S256W, N018K/N185R/A215R/S256W, N018K/N185R/A215V, N018K/N185R/Y209W/A215V/S256W, N018K/S256W, N018Q/G097P/N185K/A215V, N018Q/G097P/N185K/Y209W, N018Q/N185K, N018Q/N185K/S256W, N018Q/N185K/Y209W/A215I/S256W, N018Q/N185K/Y209W/A215V, N018Q/N185K/Y209W/A215V/S256W, N018Q/N185Q/A215V, N018Q/N185Q/S256W, N018Q/N185Q/Y209W/S256W, N018Q/N185R/A215R, N018Q/N185R/S256W, N018R/G097P/N185Q/A215I, N018R/G097P/Y209W/A215V, N018R/G097P/Y209W/S256W, N018R/N185K/A215V/S256W, N018R/N185K/S256W, N018R/N185Q/A215V/S256W, N018R/N185Q/Y209W/S256W, N018R/N185R/A215R, N018R/N185R/S256W, N018R/N185R/Y209W/S256W, N018R/Y209W/A215V/S256W, N018R/Y209W/S256W, N043E/S101E, N043E/S101E/N248K, N043E/S101F, N043E/S101N/N117Y, N043E/S101V, N043E/S101Y/N117I/N248K, N043E/S101Y/N117Y/N248K, N043F/N117I, N043F/S101E/N117I/N248K, N043F/S101R, N043I/N117Y, N043I/S101E/N248I, N043I/S101F/N117Y/N248K, N043I/S101N1N117Y/N248K, N043I/S101R/N248K, N043I/S101Y1N117I/N248I, N043I/S101Y/N117Y/N248K, N043 S/N117Y/N248K, N043 S/N248I, N043 S/S101E, N043 S/S101E/N117I/N248I, N043 S/S101E/N117Y/N248K, N043 S/S101E/N248K, N043 S/S101F, N043 S/S101F/N248K, N043 S/S101N/N117I, N043 S/S101R/N117I/N248K, N043 S/S101V/N117I/N248I, N043 S/S101Y, N043 S/S101Y/N117I, N043 S/S101Y/N117Y, N043 S/S101Y/N248K, N043V/N117Y/N248I, N043V/N248I, N043V/N248K, N043V/S101E/N117I/N248K, N043V/S101E/N248I, N043V/S101F, N043V/S101F/N117I, N043V/S101F/N117Y, N043V/S101F/N248K, N043V/S101R/N117I/N248K, N043V/S101T/N248K, N043V/S101V/N117I, N043V/S101V/N248K, N043V/S101Y, N043V/S101Y/N117I, N043V/S101Y/N117I/N248K, N185K/A215V, N185K/Y209W/S256W, N185Q/A215R/S256W, N185Q/S256W, N185R/S256W, N185R/Y209W/A215I, N185R/Y209W/S256W, N185V/Y209W, P052I/M119F, P052I/M119F/S144T/N185R/Y209W, P052I/M119F/S144V/Y209W, P052I/M119F/S144Y/N185R, P052I/M119F/S144Y/N185R/Y209W/S256I, P052I/M119F/Y209W/S256I, P052I/N185R/Y209W/S256I, P052I/N185V/Y209W, P052I/N185V/Y209W/S256I, P052I/S144T, P052I/S144T/N185R/Y209W/S256T, P052I/S144T/N185V/Y209W/S256T, P052I/S144T/S256I, P052I/S144T/Y209W/S256I, P052I/S144T/Y209W/S256T, P052I/S144V/Y209W, P052I/S144Y/N185R/Y209W, P052I/S144Y/N185V, P052I/S144Y/N185V/S256T, P052I/S144Y/S256T, P052I/S144Y/Y209W/S256I, P052V/M119F/N185V/Y209W/S256I, P052V/M119F/S144T, P052V/M119F/S144T/N185R/S256T, P052V/M119F/S144T/N185R/Y209W, P052V/M119F/S144T/S256T, P052V/M119F/S144T/Y209W, P052V/M119F/S144V/N185V/S256I, P052V/N185V/Y209W/S256I, P052V/S144T, P052V/S144T/N185V/Y209W/S256I, P052V/S144T/N185V/Y209W/S256T, P052V/S144T/Y209W/S256I, P052V/S144V/N185R/Y209W/S256T, P052V/S144V/N185V/Y209W, P052V/S144V/Y209W/S256I, P052V/S144W/N185R, P052V/S144W/N185V/Y209W, P052V/S144W/Y209W, P052V/S144Y/S256T, P052V/S144Y/Y209W, P052V/S256T, Q109A/G118S, Q109D/G118V/G127H, Q109H/G118S/S132P, Q109H/G127R, Q109I/G118R, 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S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S024W/S099T/S132N, S087N/G118V/S128L/P129Q/S130A/S099K, S087N/G118V/S128L/P129Q/S130A/S099K/S103N, S087N/G118V/S128L/P129Q/S130A/S099K/S103P, S087N/G118V/S128L/P129Q/S130A/S099K/S132H, S087N/G118V/S128L/P129Q/S130A/S099Q, S087N/G118V/S128L/P129Q/S130A/S099Q/S103N, S087N/G118V/S128L/P129Q/S130A/S099Q/S103P, S087N/G118V/S128L/P129Q/S130A/S099Q/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S099Q/S132H, S087N/G118V/S128L/P129Q/S130A/S099T/G127Q, S087N/G118V/S128L/P129Q/S130A/S099T/S103N, S087N/G118V/S128L/P129Q/S130A/S099T/S103N/S132H, S087N/G118V/S128L/P129Q/S130A/S099T/S103P, S087N/G118V/S128L/P129Q/S130A/S099T/S103P/S132H, S087N/G118V/S128L/P129Q/S130A/S099T/S132H, S087N/G118V/S128L/P129Q/S130A/S101E, S087N/G118V/S128L/P129Q/S130A/S101E/N248K, S087N/G118V/S128L/P129Q/S130A/S101F/N248K, S087N/G118V/S128L/P129Q/S130A/S101N/N248K, S087N/G118V/S128L/P129Q/S130A/S101R/N248K, S087N/G118V/S128L/P129Q/S130A/S101Y, S087N/G118V/S128L/P129Q/S130A/S103N/S132H, S087N/G118V/S128L/P129Q/S130A/S103P, S087N/G118V/S128L/P129Q/S130A/S144T/N185R, S087N/G118V/S128L/P129Q/S130A/S144T/N185R/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/N185R/Y209W, S087N/G118V/S128L/P129Q/S130A/S144T/N185R/Y209W/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/N185V/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/N185V/Y209W, S087N/G118V/S128L/P129Q/S130A/S144T/S256I, S087N/G118V/S128L/P129Q/S130A/S144T/Y209W/S256I, S087N/G118V/S128L/P129Q/S130A/S144V, S087N/G118V/S128L/P129Q/S130A/S144V/N185R/S256I, S087N/G118V/S128L/P129Q/S130A/S144V/N185R/Y209W, S087N/G118V/S128L/P129Q/S130A/S144V/N185V, S087N/G118V/S128L/P129Q/S130A/S144V/N185V/S256I, S087N/G118V/S128L/P129Q/S130A/S144V/N185V/Y209W, S087N/G118V/S128L/P129Q/S130A/S144V/Y209W/S256I, S087N/G118V/S128L/P129Q/S130A/S144V/Y209W/S256T, S087N/G118V/S128L/P129Q/S130A/S144W/Y209W, S087N/G118V/S128L/P129Q/S130A/S144Y/N185R, S087N/G118V/S128L/P129Q/S130A/S144Y/N185R/Y209W/S256T, S087N/G118V/S128L/P129Q/S130A/S144Y/N185V/Y209W, S087N/G118V/S128L/P129Q/S130A/S164I, S087N/G118V/S128L/P129Q/S130A/S164Q, S087N/G118V/S128L/P129Q/S130A/V104I, S087N/G118V/S128L/P129Q/S130A/V104I/L126F/S164F, S087N/G118V/S128L/P129Q/S130A/V104I/L126F/S164I, S087N/G118V/S128L/P129Q/S130A/V104I/S164I, S087N/G118V/S128L/P129Q/S130A/V104I/S164Q, S087N/G118V/S128L/P129Q/S130A/Y209W, S087N/G118V/S128L/P129Q/S130A/Y209W/A215I/S256W, S087N/G118V/S128L/P129Q/S130A/Y209W/S256W, S099H/S103I/G118N, S099K/G118R, S099K/S103N/G118T, S099K/S103P/G118L/S132N, S099K/S103P/G127Q, S099K/S103P/S132H, S099K/S103R/G118A/S132P, S099N/S132R, S099Q/G118F/G127R, S099Q/G118T/G127Q, S099Q/S103N/G118R/S132N, S099Q/S103N/G118T, S099Q/S103N/G118T/S132H, S099Q/S103N/S132H, S099Q/S103P/G118R, S099Q/S103P/G118R/S132N, S099Q/S103P/S132H, S099Q/S132R, S099T/G118F, S099T/G118R/S132H, S099T/G118T/S132H, S099T/G118T/S132N, S099T/S103P, S099T/S103P/G118L/S132H, S101E/N117I, S101E/N117Y/N248K, S101F/N117I/N248I, S101F/N117Y/N248I, S101F/N117Y/N248K, S101F/N248K, S101R/N117I/N248K, S101R/N117Y, S101R/N248K, S101T/N117Y/N248K, S101T/N248I, S101V/N248K, S101Y/N117I/N248K, S101Y/N117Y, S101Y/N248K, S103G/G118I/G127E/S132E, S103L/S132E, S103R/S132P, S144T/N185R, S144T/N185R/Y209W/S256I, S144T/N185R/Y209W/S256T, S144T/N185V, S144V/N185R/S256I, S144V/N185R/Y209W, S144V/N185R/Y209W/S256T, S144V/S256I, S144V/Y209W, S144W/N185R/Y209W/S256T, S144W/N185V/Y209W/S256I, S144W/S256I, S144Y/Y209W/S256I, V104E/L126I/S164L, wherein the positions 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 of a  Bacillus  subtilisin, wherein said subtilisin variant is a mature form having proteolytic activity and comprising substitutions selected from: TS87N/G118V/S128L/P129Q/S130A/S24W/S101L/Q109K, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/172V/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/Q109L/N185R/A215R, S87R/G118R/S128L/P129Q/S130A/S101K/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S24W/S101L/Q109L, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/172V/S101L/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109R/S188D/T213E/N248R, S87R/G118R/S128L/P129Q/S130A/172V/S164I/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D, S87N/G118V/S128L/P129Q/S130A/S24W/S101Y/Q109K, S87N/G118V/S128L/P129Q/S130A/N43S/P52V/S101R/Q109R, S87N/G118V/S128L/P129Q/S130A/172V/S101Y/Q109R/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/Q109L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/172V/Q109R/S164I/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D/S101L, S87N/G118V/S128L/P129Q/S130A/S24W/S101Y/Q109L, S87N/G118V/S128L/P129Q/S130A/N76D/S164I, S87R/G118R/S128L/P129Q/S130A/N76D/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109L/S188D/N248R, S87R/G118R/S128L/P129Q/S130A/V118R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N76D/S101M, S87N/G118V/S128L/P129Q/S130A/N18K/N76D/G97P/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/172V/S101L/S164I, S87R/G118R/S128L/P129Q/S130A/S101L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/S101Y/Q109L/S188D/T213E/N248R, S87R/G118R/S128L/P129Q/S130A/Q109L/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/S164I, S87N/G118V/S128L/P129Q/S130A/S101Y/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/S101L/Q109L/S164I, S87R/G118R/S128L/P129Q/S130A/S101V/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101Y/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101V/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/T213E/N248R, S87N/G118V/S128L/P129Q/S130A/I72V/S101Y/Q109L/S164I, S87R/G118R/S128L/P129Q/S130A/S101H/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/Q109R/N185R/A215R, S87N/G118V/S128L/P129Q/S130A/I72V/S101H/S164I, S87N/G118V/S128L/P129Q/S130A/S101L/Q109R/S188D/N248R, S87N/G118V/S128L/P129Q/S130A/N18K/G97P/S101L/Q109L/N185R/A215R, S87R/G118R/S128L/P129Q/S130A/S101M/S188D/N248R, N76D/S87R/G118R/S128L/P129Q/S130A/S188D, N76D/S87R/G118R/S128L/P129Q/S130A/S188D/N248K, S87N/G118V/G127S/S128L/P129Q/S130A, S87N/S101H/G118V/S128L/P129Q/S130A, S87N/S101K/G118V/S128L/P129Q/S130A, S87N/S101V/G118V/S128L/P129Q/S130A, S87N/S101Y/G118V/S128L/P129Q/S130A, S87N/S101L/G118V/S128L/P129Q/S130A, I72V/S87N/G118V/S128L/P129Q/S130A/S164I, wherein the positions are numbered by correspondence with the amino acid sequence of  B. amyloliquefaciens  subtilisin BPN′ set forth as SEQ ID NO:1. 
     
     
         24 . An isolated nucleic acid encoding the subtilisin variant set forth in  claim 19 . 
     
     
         25 . An isolated nucleic acid encoding the subtilisin variant set forth in  claim 22 . 
     
     
         26 . An expression vector comprising the nucleic acid of  claim 19 . 
     
     
         27 . An expression vector comprising the nucleic acid of  claim 22 . 
     
     
         28 . A host cell comprising the expression vector of  claim 26 . 
     
     
         29 . A host cell comprising the expression vector of  claim 27 . 
     
     
         30 . A cleaning composition comprising the subtilisin variant set forth in  claim 19 . 
     
     
         31 . A cleaning composition comprising the subtilisin variant set forth in  claim 22 . 
     
     
         32 . A cleaning composition comprising the subtilisin variant set forth in  claim 23 . 
     
     
         33 - 38 . (canceled) 
     
     
         39 . The cleaning composition of  claim 30 , wherein said composition comprises a liquid, gel, tablet, powder and/or granule detergent. 
     
     
         40 . The cleaning composition of  claim 31 , wherein said composition comprises a liquid, gel, tablet, powder and/or granule detergent. 
     
     
         41 . The cleaning composition of  claim 32 , wherein said composition comprises a liquid, gel, tablet, powder and/or granule detergent. 
     
     
         42 - 47 . (canceled) 
     
     
         48 . The cleaning composition of  claim 30 , wherein said cleaning composition is selected from laundry detergents and dish detergents. 
     
     
         49 . The cleaning composition of  claim 31 , wherein said cleaning composition is selected from laundry detergents and dish detergents. 
     
     
         50 . The cleaning composition of  claim 32 , wherein said cleaning composition is selected from laundry detergents and dish detergents. 
     
     
         51 - 56 . (canceled) 
     
     
         57 . The cleaning composition of  claim 30 , comprising a laundry detergent. 
     
     
         58 . The cleaning composition of  claim 57 , wherein said laundry detergent is a heavy duty detergent. 
     
     
         59 . The cleaning composition of claim  56 , comprising a dish detergent selected from hand dishwashing and automatic dishwashing detergents. 
     
     
         60 . The cleaning composition of  claim 30 , further comprising at least one bleaching agent. 
     
     
         61 . The cleaning composition of  claim 30 , wherein said cleaning composition is a phosphate-free or phosphate-containing detergent. 
     
     
         62 . The cleaning composition of any of  claim 30 , wherein said cleaning composition is a cold water detergent. 
     
     
         63 . The cleaning composition of any of  claim 30 , further comprising at least one additional enzyme. 
     
     
         64 . The cleaning composition of  claim 63 , wherein said at least one additional enzyme is selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolasess, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ß-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. 
     
     
         65 . A method for cleaning comprising providing an item to be cleaned and a composition comprising the cleaning composition set forth in  claim 30 , and contacting said item with said composition, under conditions effective to provide cleaning of said item. 
     
     
         66 . The method of  claim 65 , further comprising the step of rinsing said item after contacting said item with said cleaning composition. 
     
     
         67 . The method of  claim 65 , wherein said item to be cleaned comprises dishware. 
     
     
         68 . The method of  claim 65 , wherein said item to be cleaned comprises fabric.

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