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US 2010/0304464 A1Published application
Stable, functional chimeric cellobiohydrolases
AssigneeCalifornia Institute of Technology
InventorsFrances H. Arnold, Pete Heinzelman, Jeremy Minshull, Sridhar Govindarajan, Alan Villalobos
Life of this patent
Estimating the term…
Abstract
The present disclosure relates to CBH II chimera fusion polypeptides, nucleic acids encoding the polypeptides, and host cells for producing the polypeptides.
Claims15
A chimeric polypeptide comprising at least two domains from two different parental cellobiohydrolase II (CBH II) polypeptides, wherein the domains comprise from N- to C-terminus:
- (segment 1)-(segment 2)-(segment 3)-(segment 4)-(segment 5)-(segment 6)-(segment 7)-(segment 8);
- wherein:
- segment 1 comprises a sequence that is at least 50-100% identical to amino acid residue from about 1 to about x1 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 2 comprises a sequence that is at least 50-100% identical to amino acid residue x1 to about x2 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 3 comprises a sequence that is at least 50-100% identical to amino acid residue x2 to about x3 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 4 comprises a sequence that is at least 50-100% identical to amino acid residue x3 to about x4 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 5 comprises a sequence that is at least 50-100% identical to about amino acid residue x4 to about x5 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 6 comprises a sequence that is at least 50-100% identical to amino acid residue x5 to about x6 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 7 comprises a sequence that is at least 50-100% identical to amino acid residue x6 to about x7 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- and segment 8 comprises a sequence that is at least 50-100% identical to amino acid residue x7 to about x8 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- wherein x1 is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6;
- x2 is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6;
- x3 is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6;
- x4 is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6;
- x5 is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6;
- x6 is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6;
- x7 is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4 or SEQ ID NO:6;
- and x8 is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6
- wherein the chimeric polypeptide has cellobiohydrolase activity and improved thermostability and/or pH stability compared to a CBH II polypeptide comprising SEQ ID NO:2, 4, or 6.
16 more elements
A method of generating a thermostable chimeric cellobiohydrolase polypeptide, comprising recombining segments from at least 3 parental cellobiohydrolase polypeptide wherein the chimeric polypeptide comprises from N- to C-terminus 8 segments wherein:
- segment 1 comprises a sequence that is at least 50-100% identical to amino acid residue from about 1 to about x1 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 2 comprises a sequence that is at least 50-100% identical to amino acid residue x1 to about x2 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 3 comprises a sequence that is at least 50-100% identical to amino acid residue x2 to about x3 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 4 comprises a sequence that is at least 50-100% identical to amino acid residue x3 to about x4 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 5 comprises a sequence that is at least 50-100% identical to about amino acid residue x4 to about x5 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 6 comprises a sequence that is at least 50-100% identical to amino acid residue x5 to about x6 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- segment 7 comprises a sequence that is at least 50-100% identical to amino acid residue x6 to about x7 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- and segment 8 comprises a sequence that is at least 50-100% identical to amino acid residue x7 to about x8 of SEQ ID NO:2 (“1”), SEQ ID NO:4 (“2”) or SEQ ID NO:6 (“3”);
- wherein x1 is residue 43, 44, 45, 46, or 47 of SEQ ID NO:2, or residue 42, 43, 44, 45, or 46 of SEQ ID NO:4 or SEQ ID NO:6;
- x2 is residue 70, 71, 72, 73, or 74 of SEQ ID NO:2, or residue 68, 69, 70, 71, 72, 73, or 74 of SEQ ID NO:4 or SEQ ID NO:6;
- x3 is residue 113, 114, 115, 116, 117 or 118 of SEQ ID NO:2, or residue 110, 111, 112, 113, 114, 115, or 116 of SEQ ID NO:4 or SEQ ID NO:6;
- x4 is residue 153, 154, 155, 156, or 157 of SEQ ID NO:2, or residue 149, 150, 151, 152, 153, 154, 155 or 156 of SEQ ID NO:4 or SEQ ID NO:6;
- x5 is residue 220, 221, 222, 223, or 224 of SEQ ID NO:2, or residue 216, 217, 218, 219, 220, 221, 222 or 223 of SEQ ID NO:4 or SEQ ID NO:6;
- x6 is residue 256, 257, 258, 259, 260 or 261 of SEQ ID NO:2, or residue 253, 254, 255, 256, 257, 258, 259 or 260 of SEQ ID NO:4 or SEQ ID NO:6;
- x7 is residue 312, 313, 314, 315 or 316 of SEQ ID NO:2, or residue 309, 310, 311, 312, 313, 314, 315 or 318 of SEQ ID NO:4 or SEQ ID NO:6;
- and x8 is an amino acid residue corresponding to the C-terminus of the polypeptide have the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6;
- screening the chimeric polypeptide for the ability to hydrolyze cellulose at a temperature of about 63° C.
14 more elements
Dependencies are inferred from the claim text.