Variant lipolytic enzymes
The inventors have developed improved polypeptides by substituting or deleting specified amino acids in fungal lipolytic enzymes. More particularly, the polypeptides result in a reduction of dough stickiness when they are added to a dough. The polypeptides may particularly have activity on polar lipids.
1. A variant polypeptide which has hydrolytic activity towards an ester bond in a polar lipid and comprises an amino acid sequence which has at least 95% identity to SEQ ID NO: 1 or 2 and comprises a substitution at position 29, 33, 83 or 255 with a different amino acid than originally present in the sequence, wherein (a) the substitution at position 29 is with a different amino acid; (b) the substitution at position 33 is with a different amino acid; (c) the substitution at position 83 is with Asp, Gln, or Met; (d) the substitution at position 255 is with Gln, Ile, Leu, or Val; and wherein each position corresponds to the position of the amino acid sequence of SEQ ID NO: 1.
2. The polypeptide of claim 1 , wherein the polypeptide has phospholipase activity or galactolipase activity (EC 3.1.1.26).
3. The polypeptide of claim 1 , which comprises an amino acid sequence which has at least 95% identity to SEQ ID NO: 1.
4. The polypeptide of claim 1 , which comprises an amino acid sequence which has at least 95% identity to SEQ ID NO: 2.
5. The polypeptide of claim 3 , which comprises a substitution at position 29.
6. The polypeptide of claim 5 , wherein the substitution at position 29 is with Pro.
7. The polypeptide of claim 3 , which comprises a substitution at position 33.
8. The polypeptide of claim 7 , wherein the substitution at position 33 is with Asn.
9. The polypeptide of claim 3 , which comprises a substitution at position 83 with Asp, Gln, or Met.
10. The polypeptide of claim 3 , which comprises a substitution at position 255.
11. The polypeptide of claim 10 , wherein the substitution at position 255 is with Val.
12. The polypeptide of claim 3 , which comprises the sequence of amino acids 1-272, 1-273, 1-274 or 1-286 of SEQ ID NO: 1 and comprises:
A29P,
K33N
A29P +I83C,
A29P +I83F,
A29P +I83L,
K33N +I83N,
K33N +I83L +A255V,
K29P +I83T,
K33N +I83L,
K33N +I83N,
K33N +I83Q,
K33N +I83R,
K33N +I83S,
K33N +I83W, or
K33N +270VASLGDDTEAPRASTRGPP.
13. The polypeptide of claim 1 , which further comprises
a substitution at position 62 with A or T;
a substitution at position 91 with G or T;
a substitution at position 99 with D, F, G, or S;
a substitution at position 99 with E;
a substitution at position 158 with G;
a substitution at position 240 with D;
a substitution at position 247 with S;
a substitution at position 248 with D;
a substitution at position 249 with K or R;
a substitution at position 250 with K or T;
a substitution at position 251 with T;
a substitution at position 252 with F;
a substitution at position 253 with M or R;
a substitution at position 254 with S, W, or Y;
a substitution at position 255 with L;
a substitution at position 257 with G;
a substitution at position 260 with H or C;
a substitution at position 263 with G;
a substitution at position 264 with L;
a substitution at position 265 with I;
a substitution at position 266 with A, G, or S;
a substitution at position 267 with T;
a substitution at position 269 with L; and/or
is truncated after position 269.
14. A method of producing a dough, comprising: adding a variant lipolytic enzyme of claim 1 to a dough.
15. The method of claim 14 , Wherein said method further comprises baking the dough.