Patent ID: 6455295
Filing Date: 2002-09-24
Classification: A61K,A61Q,C11D,C12N

Abstract:
An isolated subtilisin Carlsberg variant having a modified amino acid sequence of the Subtilisin Carlsberg wild-type amino acid sequence set forth in SEQ ID NO:1, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region, a fifth loop region and a sixth loop region; wherein the modified amino acid sequence comprises a substitution at one or more position in one of the loop regions; whereinA. when the substitution occurs in the first loop region, the substitution occurs at one of positions 58, 60, 61, 62, 64 or 65; wherein a. when a substitution occurs at position 58, the substituting amino acid is Asn, Gln, Pro or Ser; b. when a substitution occurs at position 60, the substituting amino acid is Asn, Gln, Pro or Ser; c. when a substitution occurs at position 61, the substituting amino acid is Gln or Ser; d. when a substitution occurs at position 62, the substituting amino acid is Asn, Gln, Glu, Pro or Ser; e. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and f. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; B. when the substitution occurs in the second loop region, the substitution occurs at one of positions 94, 95, 96, 99, 101, or 105; wherein a. when a substitution occurs at position 94, the substituting amino acid is Ala, Gln, Gly, His, Met, Pro or Ser; b. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; c. when a substitution occurs at position 96, the substituting amino acid is Gln; d. when a substitution occurs at position 99, the substituting amino acid is Asn, Gln, Pro or Ser; e. when a substitution occurs at position 101, the substituting amino acid is Asn, Gln, Pro or Ser; and f. when a substitution occurs at position 105, the substituting amino acid is Asn, Gln, Pro or Ser; C. when the substitution occurs in the third loop region, the substitution occurs at one of positions 127, 130, 131 or 132; wherein a. when a substitution occurs at position 127, the substituting amino acid is Asn, Gln or Pro; b. when a substitution occurs at position 130, the substituting amino acid is Asn, Gln, Pro or Ser; c. when a substitution occurs at position 131, the substituting amino acid is Glu; and d. when a substitution occurs at position 132, the substituting amino acid is Asn, Gln, Pro or Ser; D. when the substitution occurs in the fourth loop region, the substitution occurs at one of positions 153, 156, 157, 159, 161, 162, 163, or 166; wherein a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; b. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu or Pro; c. when a substitution occurs at position 157, the substituting amino acid is Gln; d. when a substitution occurs at position 159, the substituting amino acid is Asn, Gln, Pro or Ser; e. when a substitution occurs at position 161, the substituting amino acid is Asn, Gln, Pro or Ser; f. when a substitution occurs at position 162, the substituting amino acid is Gln; g. when a substitution occurs at position 163, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; and h. when a substitution occurs at position 166, the substituting amino acid is His, Ile, Leu, Met or Pro; E. when the substitution occurs in the fifth loop region, the substitution occurs at position 189; wherein the substituting amino acid is Asp or Glu; and F. when the substitution occurs in the sixth loop region, the substitution occurs at one of positions 199, 200, 201, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 215, or 219; wherein a. when a substitution occurs at position 199, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr; b. when a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; c. when a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 204, the substituting amino acid is Ala, Cys, Gln, Gly, His, Met, Pro or Ser; e. when a substitution occurs at position 205, the substituting amino acid is His, Ile, Leu, Met or Pro; f. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu; g. when a substitution occurs at position 207, the substituting amino acid is Asn, Gln, Pro or Ser; h. when a substitution occurs at position 208, the substituting amino acid is His, Ile, Met or Pro; i. when a substitution occurs at position 209, the substituting amino acid is Asn, Gln, Gly or Ser; j. when a substitution occurs at position 210, the substituting amino acid is Asn, Gln, Pro or Ser; k. when a substitution occurs at position 211, the substituting amino acid is Gln or Ser; l. when a substitution occurs at position 212, the substituting amino acid is Asn, Gln, Pro or Ser; m. when a substitution occurs at position 213, the substituting amino acid is His, Ile, Leu, Met or Pro; n. when a substitution occurs at position 215, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; and o. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; whereby the subtilisin Carlsberg variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type subtilisin Carlsberg and wherein an amino acid substitution at subtilisin Carlsberg positions 58, 94, 95, 96, 99, 101, 127, 153, 156, 157, 159, 161 to 163, 166, 212 and 213 is combined with at least one further substitution at a corresponding position selected from subtilisin Carlsberg positions 59, 60, 64, 65, 104, 105,129, 131, 132, 187,189, 199 to 202, 204, 206, 209 to 211, 215 and 219.