| 主权项 |
1. A method for making an isolated chymosin polypeptide variant comprising:
(a) producing and isolating a chymosin polypeptide variant of a parent polypeptide having chymosin activity, wherein the amino acid sequence of the variant differs from the amino acid sequence of the parent by having an alteration comprising a substitution, a deletion or an insertion in at least one amino acid position of the parent sequence corresponding to any of positions 70; 75; 77; 79; 90; 102; 103; 108; 114; 117; 120; 124; 134; 154; 156; 163; 212; 222; 223; 224; 238; 246; 256; 261; 309; 320; 324; 326; 331; 336; 346; 361; 367 and 379, and/or one or more substitutions selected from K279V; L280; F281; R300D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W; and R312D,E,S,T,N,Q,C,U,G,P,A,V,I,L,M,F,Y,W, wherein the corresponding position in the parent sequence is determined by alignment of the parent sequence with SEQ ID NO: 1 (bovine chymosin), thereby obtaining the isolated chymosin polypeptide variant, wherein the variant has chymosin activity; wherein: the amino acid sequence of the parent polypeptide has at least 65% sequence identity with the amino acid sequence from position 59 to position 381 of SEQ ID NO: 1 (the mature bovine chymosin polypeptide); with the proviso that the isolated chymosin polypeptide variant is NOT a specific variant having substitutions as compared to SEQ ID NO:1 selected from the group consisting of: Q246E+G309D+S329P+D337E; R125Q+G128N+H204R+Q246E+S284T; Y185F+R213Q+Q246E; V261A+V263I+G309W+L311I+Y326F; G128D+L188I+Y326F; G128N+R312S+S313Y+Y326F; G128N+R312S+S313Y+Y326F; D117N+V261A+R312S; D216S+L224V+V263I+F281V+G309D; Y79S+L224V+L311I; and R119S+L224V+T297S. |
