Patent ID: 8114635

Claim:
A method for obtaining a variant of a microbial trypsin, comprising: (a) introducing the specific combination of modifications as follows: substitutions at positions corresponding to positions 144, 193, 198, 201, 218, 223, 227, 228, 229, 230, and 231 of amino acids 25 to 248 of SEQ ID NO: 2; deletions at positions corresponding to positions 192, 197, and 226 of amino acids 25 to 248 of SEQ ID NO: 2; and an insertion between positions corresponding to positions 224 and 225 of amino acids 25 to 248 of SEQ ID NO: 2; wherein, the variant has chymotrypsin activity and (i) has at least 90% identity to amino acids 25 to 248 of SEQ ID NO: 2; or (ii) is encoded by a nucleotide sequence which hybridizes under at least medium-high stringency conditions with nucleotides 202 to 801 of SEQ ID NO: 1 or the complete complementary strand thereto, wherein medium-high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 35% formamide followed by washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.; and (b) expressing the variant in a host cell, and (c) recovering the variant.