Multiply-substituted protease variants

Abstract

Novel enzyme variants including protease variants derived from the DNA sequences of naturally-occurring or recombinant non-human proteases are disclosed. The variant proteases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant protease to generate the substitution of a plurality of amino acid residues in the amino acid sequence of a precursor protease. Such variant proteases have properties which are different from those of the precursor protease, such as altered wash performance. The substituted amino acid residue correspond to positions 27, 45, 170, 181, 251 and 271 of Bacillus amyloliquefaciens subtilisin. Additional variants comprising at least one additional substitution at a position selected from 1, 14, 49, 61, 87, 100, 102, 118, 128, 204 and 258 of Bacillus amyloliquefaciens subtilisin are also described.

1 - 10 . (canceled) 
     
     
         11 . A subtilisin variant of a precursor subtilisin, said subtilisin variant comprising one or more modifications at a charged amino acid residue position, in the sequence set forth in SEQ ID NO:3, said subtilisin variant being characterized by having the same net electrostatic charge as said precursor subtilisin, wherein said subtilisin variant comprises an amino acid sequence having a substitution at the residue position equivalent to residue position 251, and further comprising a substitution at one or more positions corresponding to 1, 14, 49, 61, 87, 100, 102, 118, 128, 204, and 258. 
     
     
         12 . The subtilisin variant of  claim 11 , further comprising an additional substitution at position corresponding to position 1, of  Bacillus amyloliquefaciens  subtilisin as set forth in SEQ ID NO:3. 
     
     
         13 . A DNA encoding the protease variant of  claim 11 . 
     
     
         14 . An expression vector comprising the DNA of  claim 13 . 
     
     
         15 . A host cell comprising the expression vector of  claim 14 . 
     
     
         16 . A cleaning composition comprising the protease variant of  claim 11 .