Compositions and methods comprising protease variants

Abstract

The present invention provides protease variants, compositions comprising protease variants, and methods of using such protease variants and compositions.

1 . An isolated protease variant of the parent subtilisin protease having the sequence of SEQ ID NO:2, wherein the variant comprises an amino acid sequence comprising three amino acid substitutions selected from the group consisting of X024G/R, X053G, X078N, X101N, X128A/S, and X217L/Q, wherein the variant has proteolytic activity, at least 85% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6, enhanced proteolytic activity and/or cleaning activity compared to the proteolytic activity of the BPN′ protease having the sequence of SEQ ID NO:2 or enhanced proteolytic activity compared to the proteolytic activity of the protease having the sequence of SEQ ID NO:4 or 6, and each amino acid position of the variant is numbered by correspondence to an amino acid position in the amino acid sequence of SEQ ID NO:2 as determined by alignment of the amino acid sequence of the variant with SEQ ID NO:2. 
     
     
         2 . The variant of  claim 1 , wherein the variant amino acid sequence comprises four or five amino acid substitutions selected from the group consisting of X024G/R, X053G, X078N, X101N, X128A/S, and X217L/Q. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The variant of  claim 1 , wherein the variant amino acid sequence comprises amino acid substitutions
 X097A+X128A/S+X217L/Q or G097A+G128A/S+Y217L/Q.   
     
     
         7 - 8 . (canceled) 
     
     
         9 . The variant of  claim 6 , wherein the variant amino acid sequence further comprises a set of amino acid substitutions selected from the group consisting of:
 A088T+N109G+A116T+G131H+N243V+L257G,   S033T+N076D,   S009T+N109G+K141R+N243V,   S162G+K256R,   N109G+A116T,   N109G+L257G,   S162G+L257G,   N061G+N109G+N243V,   N109G+N243V+S248A,   S033T+N076D+N109G+N218S+N243V+S248N+K256R,   N109G+A116T+N243V+K256R,   A088T+N109G+A116T+G131H+N243V,   A088T+N109G,   N109G+N243V,   T158S+L257G,   N061S+N109G+N243V,   P040A+N109G+N243V+S248N+K256R,   S009T+S018T+Y021N+N109G+K141R,   A088T+N109G+A116T+T158S+N243V+K256R,   A088T+N109G+A116T+T158S+N218S+L257G,   N109G+K256R,   N109G+N243V+K256R,   S063G+K256R,   S063G+N109G,   S063G,   S063G+N076D,   S033T+N076D+N218S, and   N076D+N218S.   
     
     
         10 - 16 . (canceled) 
     
     
         17 . The variant of  claim 1 , wherein the parent protease has at least 80% or 85% sequence identity to the  B. amyloliquefaciens  subtilisin protease BPN′ having the amino acid sequence of SEQ ID NO:2. 
     
     
         18 - 19 . (canceled) 
     
     
         20 . An isolated protease variant of the parent subtilisin protease having the sequence of SEQ ID NO:2, wherein the protease variant has proteolytic activity, at least 85% sequence identity to SEQ ID NO:2, enhanced proteolytic activity and/or cleaning activity compared to the proteolytic activity of the protease set forth in SEQ ID NO:2 or enhanced proteolytic activity compared to the proteolytic activity of the protease having the sequence of SEQ ID NO:4 or 6, and comprises an amino acid sequence comprising an alteration at three amino acid positions corresponding to amino acid positions of SEQ ID NO:2 selected from the group consisting of positions 24, 53, 78, 97, 101, 128, and 217, wherein the at least one alteration is independently
 (i) an insertion of one or more amino acid residues upstream or downstream of the amino acid residue which occupies the position,   (ii) a deletion of the amino acid residue which occupies the position, or   (iii) a substitution of the amino acid residue which occupies the position with a different amino acid residue,   wherein each amino acid position is numbered by correspondence with an amino acid position in the amino acid sequence of  Bacillus amyloliquefaciens  subtilisin protease BPN′ set forth in SEQ ID NO:2 as determined by alignment of the amino acid sequence of the variant with SEQ ID NO:2.   
     
     
         21 - 22 . (canceled) 
     
     
         23 . The variant of  claim 20 , wherein the variant comprises an amino acid sequence comprising an alteration at four, five, or six amino acid positions selected from the group consisting of positions 24, 53, 78, 97, 101, 128, and 217. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The variant of  claim 20 , wherein the variant comprises an amino acid sequence comprising an alteration at each of the amino acid positions corresponding to positions of SEQ ID NO:2 selected from the group consisting of positions 24, 53, 78, 97, 101, 128, and 217. 
     
     
         27 - 34 . (canceled) 
     
     
         35 . The variant of  claim 20 , wherein the variant comprises an amino acid sequence comprising at least three amino acid substitutions selected from the group consisting of X024G/R, X053G, X078N, X097A, X101N, X128A/S, and X217Q/L. 
     
     
         36 . The variant of  claim 23 , wherein the variant comprises an amino acid sequence comprising at least four, five, or six amino acid substitutions selected from the group consisting of X024G/R, X053G, X078N, X097A, X101N, X128A/S, and X217Q/L. 
     
     
         37 - 38 . (canceled) 
     
     
         39 . The variant of  claim 20 , wherein the variant comprises an amino acid sequence comprising the substitutions G097A, G128A/S, and Y217L/Q. 
     
     
         40 - 48 . (canceled) 
     
     
         49 . The variant of  claim 20 , wherein the parent protease has at least 80% or 85% sequence identity to the  B. amyloliquefaciens  subtilisin protease BPN′ having the amino acid sequence of SEQ ID NO:2. 
     
     
         50 - 52 . (canceled) 
     
     
         53 . The isolated variant of  claim 1 , wherein said variant comprises an amino acid sequence selected from the group consisting of:
 a) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+G131H+N243V+L257G;   b) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+S033T+N076D;   c) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+S009T+N109G+K141R+N243V;   d) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+S162G+K256R;   e) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+A116T;   f) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+L257G;   g) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+S162G+L257G;   h) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N061G+N109G+N243V;   i) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+N109G+N243V+S248A;   j) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+S033T+N076D+N109G+N218S+N243V+S248N+K256R;   k) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+A116T+N243V+K256R;   l) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+G131H+N243V;   m) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G;   n) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+N243V;   o) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+T158S+L257G;   p) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N061S+N109G+N243V;   q) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+P040A+N109G+N243V+S248N+K256R;   r) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+S009T+S018T+Y021N+N109G+K141R;   s) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+T158S+N243V+K256R;   t) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+A088T+N109G+A116T+T158S+N218S+L257G;   u) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N109G+K256R;   v) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+N109G+N243V+K256R;   w) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+S063G+K256R;   x) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+S063G+N109G;   y) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+S063G;   z) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+S063G+N076D;   aa) BPN′-S024G+S053G+S078N+S101N+G128S+Y217Q+S033T+N076D+N218S;   bb) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q+N076D+N218S; and   cc) BPN′-S024G+S053G+S078N+S101N+G128A+Y217Q, wherein each amino acid position of the variant is numbered by correspondence with an amino acid position of the sequence of SEQ NO:2.   
     
     
         54 . An isolated nucleic acid comprising a polynucleotide sequence encoding a variant of  claim 1 , or a complementary polynucleotide sequence thereof. 
     
     
         55 . An isolated nucleic acid comprising a polynucleotide sequence having at least 80% sequence identity to the polynucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, or a complementary polynucleotide sequence thereof. 
     
     
         56 . An expression vector comprising at least one nucleic acid of  claim 54 . 
     
     
         57 . (canceled) 
     
     
         58 . A recombinant host cell comprising an expression vector of  claim 56 . 
     
     
         59 - 62 . (canceled) 
     
     
         63 . A method of producing a protease variant, the method comprising cultivating a recombinant host cell of  claim 58  under conditions conducive to produce the variant. 
     
     
         64 - 66 . (canceled) 
     
     
         67 . A composition comprising a variant of  claim 1 , which composition is not a fabric and home care product. 
     
     
         68 - 73 . (canceled) 
     
     
         74 . A method for cleaning an item or surface in need of cleaning, the method comprising contacting the item or surface with a variant of  claim 1 . 
     
     
         75 - 78 . (canceled)