Patent ID: 6451574
Filing Date: 2002-09-17
Classification: A61K,A61Q,C11D,C12N

Abstract:
A Proteinase K variant having a modified amino acid sequence of the Proteinase K 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 positions in one of the loop regions; whereinA. when the substitution occurs in the first loop region, the substitution occurs at one of positions 65, 66, 68, 70 or 71; wherein a. when a substitution occurs at position 65, the substituting amino acid is Glu; b. when a substitution occurs at position 66, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; c. when a substitution occurs at position 68, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 70, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; and e. when a substitution occurs at position 71, 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 95, 96, or 106; wherein a. when a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Pro, Ser or Thr; b. when a substitution occurs at position 96, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; and c. when a substitution occurs at position 106, the substituting amino acid is Asn, Gln, Gly, Pro or Ser; C. when the substitution occurs in the third loop region, the substitution occurs at one of positions 137, 139 or 140; wherein a. when a substitution occurs at position 137, the substituting amino acid is Asp, His, Ile, Leu, Met or Pro; b. when a substitution occurs at position 139, the substituting amino acid is Asp or Glu; and c. when a substitution occurs at position 140, the substituting amino acid is Asp or Glu; D. when the substitution occurs in the fourth loop region, the substitution occurs at position 162; wherein the substituting amino acid is Asp, or Glu; E. when the substitution occurs in the fifth loop region, the substitution occurs at position 193; 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 203, 204, 205, 206, 208, 209, 210, 260, 212, 213, 214, 215, 219, or 223; wherein a. when a substitution occurs at position 203, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; b. when a substitution occurs at position 204, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser; c. when a substitution occurs at position 205, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; d. when a substitution occurs at position 206, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser; e. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; f. when a substitution occurs at position 209, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val; g. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu; h. when a substitution occurs at position 211, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; i. when a substitution occurs at position 212, the substituting amino acid is Asp or Glu; j. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val; k. when a substitution occurs at position 214, the substituting amino acid is Asn, Gln, Pro or Ser; l. when a substitution occurs at position 215, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; n. when a substitution occurs at position 219, the substituting amino acid is Asp or Glu; and o. when a substitution occurs at position 223, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser; whereby the Proteinase K variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Proteinase K and wherein amino acid substitutions at proteinase K position 162 or proteinase K positions 95 and 96 or substitutions of aspartate, cysteine and glutamate at proteinase K position 209 or 211 are combined with at least one further substitution at a corresponding position selected from the proteinase K positions: 65, 66, 68, 70, 71, 105, 106, 137, 139, 140, 191, 193, 203 to 206, 208 to 215, 219 and 223.