Subtilases

摘要

The present invention relates to methods for producing variants of a parent TY145 subtilase and of a parent BPN′ subtilase and to TY145 and BPN′ variants having altered properties as compared to the parent TY145/BPN′ subtilase.

主权项

1. A TY145 like subtilase variant which is at least 63% homologous to the sequence of SEQ ID NO:1, which variant comprises:
(1) an alteration in one or more positions located at a distance of not more than 10 Å to one of the ion-binding sites of TY145, wherein the positions, as specified in SEQ ID NO:1, located at a distance of not more than 10 Å to:
a) the Weak ion-binding site are: 154, 155, 158, 164, 165, 166, 167, 168, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 211, 220, 221, 222, 223, 224, 225, 226, 227, 228, 277, 281 and 305,b) the Near ion-binding site are: 185, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 277, 281, 299, 300, 301, 304, 305,c) the Far ion-binding site are: 193, 198, 199, 201, 202, 204, 216, 217, 219, 226, 227, 228, 229, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 and 307; (2) one or more alterations in one or more of the positions contained in the following highly mobile regions:
84, 85, 86, 87 and 88,108, 109, 110, 111, 112, 113, 114, 115, 116 and 117,141, 142, 143, 144, 145 and 146,150, 151 and 152,169, 170 and 171,200 and 201,211, 212, 213, 214, 215, 216, 217, 218, 219 and 220,242 and 243, 268, 269 and 270; (3) one or more alterations in one or more of the positions contained in the following mobile regions:
1, 2, 3, 4, 5, 6 and 7,17, 18, 19, 20, 21, 22 and 23,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 and 69,84, 85, 86, 87, 88, 89, 90, 91 and 92,107, 108, 109 and 110,239, 240, 241, 242 and 243265 and 266,wherein said alterations preferably are introduced in one or both of the regions 57-69 and 84-92; (4) an alteration in one or more of the positions contained in the following regions:
16, 17, 18, 19, 20, 21 and 22,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 and 131,40, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 and 161,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293 and 294,wherein such alterations preferably are made in one or both the regions 40-73 and 140-161, preferably in the sub-regions 65-73 and 140-150; (5) an alteration in one or more of the following positions:
35, 36, 70, 72, 106, 109, 110, 111, 112, 113, 114, 117, 139, 140, 141, 142, 143,144, 145, 147, 150, 167, 168, 169, 170, 171, 172, 173, 174, 177, 180, 207, 239, 247,148, 149, 150, 151 and 252 of SEQ ID NO:1; or (6) an alteration in one or more of the following positions: V31, V38, T79, V80, L81, V188, T254, wherein preferred variants comprise one or more of V31I, T79S and V80A.