US 6,475,765 B1 · Subtilisin DY variants having decreased adsorption and increased hydrolysis

wherein the modified amino acid sequence comprises a substitution at two or more positions in one or more of the loop regions;

wherein

A. when a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65;

wherein

a. when a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

b. when a substitution occurs at position 59, the substituting amino acid is Glu;

c. when a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

d. when a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser;

e. when a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

f. when a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

and

g. when a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

B. when a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106;

wherein

a. when a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;

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 Asp, Gln, Glu or Ser;

but when position 96 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 103, 154, 155, 165, 188, and 216;

d. when a substitution occurs at position 97, the substituting amino acid is Asp or Glu;

but when position 97 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

e. when a substitution occurs at position 98, the substituting amino acid is Asp or Glu;

but when position 98 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

f. when a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 99 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

g. when a substitution occurs at position 100, the substituting amino acid is Asp or Glu;

but when position 100 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

h. when a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 101 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103,155, 165, 188, and 216;

i. when a substitution occurs at position 102, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

but when position 102 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

j. when a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val;

k. when a substitution occurs at position 104, the substituting amino acid is Asp or Glu;

l. when a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;

and

m. when a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val;

but when position 106 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 103, 154, 155, 165, 188, and 216;

C. when a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125, 126, 127, 128, 129, 130, 131 or 132;

wherein

a. when a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;

but when position 125 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 103, 154, 155, 165, 188, and 216;

b. when a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 126 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 103, 154, 155, 165, 188, and 216;

c. when a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 127 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 103, 154, 155, 165, 188, and 216;

d. when a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser;

but when position 128 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 103, 154, 155, 165, 188, and 216;

e. when a substitution occurs at position 129, the substituting amino acid is Asp or Glu;

f. when a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

g. when a substitution occurs at position 131, the substituting amino acid is Asp or Glu;

and

h. when a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

D. when a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166;

wherein

a. when a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 153 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

b. when a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser;

c. when a substitution occurs at position 155, the substituting amino acid is Asp or Glu;

d. when a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 156 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

e. when a substitution occurs at position 157, the substituting amino acid is Asp or Glu;

but when position 157 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

f. when a substitution occurs at position 158, the substituting amino acid is Asp or Glu;

but when position 158 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

g. when a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

but when position 159 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

h. when a substitution occurs at position 160, the substituting amino acid is Asp or Glu;

but when position 160 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

i. when a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Glu or Ser;

but when position 161 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

j. when a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser;

but when position 162 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

k. when a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

but when position 163 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

l. when a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val;

but when position 164 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

m. when a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

and

n. when a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val;

but when position 166 is substituted the variant is not a double, triple, quadruple, quintuple, or sextuple mutation variant having substitutions at positions selected from the group consisting of 103, 155, 165, 188, and 216;

E. when a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190;

wherein

a. when a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr;

b. when a substitution occurs at position 187, the substituting amino acid is Asp or Glu;

c. when a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val;

d. when a substitution occurs at position 189, the substituting amino acid is Asp or Glu;

and

e. when a substitution occurs at position 190, the substituting amino acid is Asp or Glu;

and

F. when a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218 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 202, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr;

e. when a substitution occurs at position 203, the substituting amino acid is Asp or Glu;

f. when a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, or Thr;

g. when a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val;

h. when a substitution occurs at position 206, the substituting amino acid is Asp or Glu;

i. when a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

j. when a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr or Val;

k. when a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, or Ser;

l. when a substitution occurs at position 210, the substituting amino acid is Asp or Glu;

m. when a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser;

n. when a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

but when position 212 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 103, 154, 155, 165, 188, and 216;

o. when a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val;

but 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 103, 154, 155, 165, 188, and 216;

p. when a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

but when position 214 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 103, 154, 155, 165, 188, and 216;

q. when a substitution occurs at position 215, the substituting amino acid is Asp or Glu;

r. when a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val;

s. when a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser;

but wherein when the substituting amino acid at position 217 is Ser, the variant is not a double mutation variant having a substitution of Ser at a position selected from the group consisting of 130 and 165;

t. when a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser;

and

u. when a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser;

whereby the Subtilisin DY variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to wild-type Subtilisin DY and wherein glutamate and aspartate substitutions at both subtilisin DY positions 207 and 213, or substitutions at either of 207 and 213 combined with a serine substitution at position 217, are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219 and wherein glutamate and aspartate substitutions at both subtilisin DY positions 61 and 165 are combined with at least one further substitution at a corresponding position selected from subtilisin DY positions 59, 60, 64, 65, 104, 105, 129, 131, 132, 187, 189, 190, 199 to 202, 204, 206, 209 to 211, 215 and 219.

Subtilisin DY variants having decreased adsorption and increased hydrolysis

Life of this patent

Subtilisin DY variants having decreased adsorption and increased hydrolysis

Life of this patent

Abstract

Claims65