BPN′ variants having decreased adsorption and increased hydrolysis

An isolated BPN′ variant having a modified amino acid sequence of the Subtilisin BPN′ wild-type amino acid sequence set forth in SEQ ID NO:1, wherein the modified amino acid sequence comprises a substitution at two or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, wherein

• a. when a substitution occurs at position 199, the substituting amino acid is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• b. when a substitution occurs at position 200, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• c. when a substitution occurs at position 201, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
• d. when a substitution occurs at position 202, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
• e. when a substitution occurs at position 203, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• f. when a substitution occurs at position 204, the substituting amino acid is Asp or Glu,
• g. when a substitution occurs at position 205, the substituting amino acid is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• h. when a substitution occurs at position 206, the substituting amino acid is Pro, Asn, Ser, Asp, or Glu;
• i. when a substitution occurs at position 207, the substituting amino acid is Asp or Glu;
• j. when a substitution occurs at position 208, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• k. when a substitution occurs at position 209, the substituting amino acid is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• l. when a substitution occurs at position 210, the substituting amino acid is Ala, Gly, Gln, Asn, Ser, Asp or Glu;
• m. when a substitution occurs at position 21 1, the substituting amino acid is Ala, Pro, Gln, Asn, Ser, Asp or Glu;
• n. when a substitution occurs at position 212, the substituting amino acid is Gln, Ser, Asp or Glu;
• o. when a substitution occurs at position 213, the substituting amino acid is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• wherein when position 213 is substituted, the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and
• p. when a substitution occurs at position 214, the substituting amino acid is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu, wherein when position 214 is substituted, the variant is not a double mutation variant having a substitution at position 217, or a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 219;
• q when a substitution occurs at position 215, the substituting amino acid is Thr, Pro, Gln, Asn, Ser, Asp or Glu;
• wherein when position 215 is substituted, the variant is not a double mutation variant having a substitution at position 217, or is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 219;
• r. when a substitution occurs at position 216, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• s. when a substitution occurs at position 217, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• wherein when position 217 is substituted, the variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 219;
• t. when a substitution occurs at position 218, the substituting amino acid is Gln, Ser, Asp or Glu;
• wherein when the substituting amino acid at position 218 is Ser or Glu, the BPN′ variant is not a double mutation variant having substitutions at positions 208 and 218 wherein the substituting amino acid at position 208 is Asp or Glu, or at positions 214 and 218 wherein the substituting amino acid at position 214 is Asp or Glu, or a triple mutation variant having substitutions at positions 208, 214, and 218 wherein the substituting amino acid at position 208 is Asp or Glu and the substituting amino acid at position 214 is Asp or Glu;
• and wherein when the substituting amino acid at position 218 is Ser, Asp, or Glu, the BPN′ variant is not a double, triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 204, 213, 214, 215, and 217;
• u. when a substitution occurs at position 219, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
• and
• v. when a substitution occurs at position 220, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• whereby the BPN′ variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN′.

An isolated BPN′ variant having a modified amino acid sequence of the Subtilisin BPN′ wild-type amino acid sequence set forth in SEQ ID NO:1, wherein the modified amino acid sequence comprises a substitution at three or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220, wherein

• a. when a substitution occurs at position 199, the substituting amino acid is Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• b. when a substitution occurs at position 200, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• c. when a substitution occurs at position 201, the substituting amino acid is Gly, Gln, Asn, Ser, Asp or Glu;
• d. when a substitution occurs at position 202, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
• e. when a substitution occurs at position 203, the substituting amino acid is Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• f. when a substitution occurs at position 204, the substituting amino acid is Asp, or Glu;
• g. when a substitution occurs at position 205, the substituting amino acid is Leu, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• h. when a substitution occurs at position 206, the substituting amino acid is Pro, Asn, Ser, Asp, or Glu;
• i. when a substitution occurs at position 207, the substituting amino acid is Asp or Glu;
• j. when a substitution occurs at position 208, the substituting amino acid is Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• k. when a substitution occurs at position 209, the substituting amino acid is Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• l. when a substitution occurs at position 210, the substituting amino acid is Ala, Gly, Gln, Asn, Ser, Asp or Glu;
• m. when a substitution occurs at position 211, the substituting amino acid is Ala, Pro, Gln, Asn, Ser, Asp or Glu;
• n. when a substitution occurs at position 212, the substituting amino acid is Gln, Ser, Asp or Glu;
• o. when a substitution occurs at position 213, thesubstituting amino acid is Trp, Phe, Tyr, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• wherein when position 213 is substituted, the variant is not a triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 2199;
• p. when a substitution occurs at position 214, the substituting amino acid is Phe, Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• wherein when position 214 is substituted, the variant is not a triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 219;
• q. when a substitution occurs at position 215, the substituting amino acid is Thr, Pro, Gln, Asn, Ser, Asp or Glu;
• wherein when position 215 is substituted, the variant is not a triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 219;
• r. when a substitution occurs at position 216, the substituting amino acid is His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu;
• s. when a substitution occurs at position 217, the substituting amino acid is Leu, Ile, Val, Met, Cys, Ala, His, Thr, Pro, Gly, Gln, Asn, Ser, Asp or Glu:
• wherein when position 217 is substituted, the variant is not a triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 206, 208, 213, 214, 218, and 219;
• t. when a substitution occurs at position 218, the substituting amino acid is Gln, Ser, Asp or Glu;
• wherein when the substituting amino acid at position 218 is Ser or Glu, the BPN′ variant is not a triple mutation variant having substitutions at positions 208, 214, and 218, wherein the substituting amino acid at position 208 is Asp or Glu and the substituting amino acid at position 214 is Asp or Glu;
• wherein the variant is not a quadruple mutation variant having substitutions at positions 208, 214, and 219;
• and wherein when the substituting amino acid at position 218 is Ser, Asp, or Glu, the BPN′ variant is not a triple, quadruple, quintuple, sextuple, or septuple mutation variant having substitutions at positions selected from the group consisting of 204, 213, 214, 215, and 217;
• u. when a substitution occurs at position 219, the substituting amino acid is Pro, Gln, Asn, Ser, Asp or Glu;
• wherein the variant is not a triple mutation variant having substitutions at positions 208 and 214;
• and
• v. when a substitution occurs at position 220, the substituting amino acid is Pro, Gly, Gln, Asn, Ser Asp or Glu;
• whereby the BPN′ variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin BPN′.