Microbial Trypsin Mutants having Chymotrypsin Activity And Nucleic Acids Encoding Same

Abstract

The present invention relates to microbial trypsin variants having chymotrypsin-like activity, comprising: (a) a one or more substitutions corresponding to positions 144, S193, 198, 201, 218, 223, 227, 228, 229, 230, and 231 of amino acids 25 to 248 of SEQ ID NO: 2, (b) one or more deletions corresponding to positions 192, 197, and 226 of amino acids 25 to 248 of SEQ ID NO: 2; and (c) an insertion between positions corresponding to positions 224 and 225 of amino acids 25 to 248 of SEQ ID NO: 2. The present invention further relates to nucleotide sequences encoding microbial trypsin variants having chymotrypsin-like activity; nucleic acid constructs, expression vectors, and recombinant host cells comprising such nucleotide sequences; and methods of producing microbial trypsin variants having chymotrypsin-like activity or a precursor thereof.

1 . A microbial trypsin variant having chymotrypsin-like activity, comprising one or more modifications selected from the group consisting of:
 (a) a substitution at one or more 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;   (b) a deletion at one or more positions corresponding to positions 192, 197, and 226 of amino acids 25 to 248 of SEQ ID NO: 2; and   (c) an insertion between positions corresponding to positions 224 and 225 of amino acids 25 to 248 of SEQ ID NO: 2;   
       wherein the microbial trypsin is (a) a polypeptide having an amino acid sequence which has at least 90% identity to amino acids 25 to 248 of SEQ ID NO: 2; or (b) a polypeptide encoded by a nucleotide sequence which hybridizes under at least high stringency conditions with nucleotides 202 to 801 of SEQ ID NO: 1 or its full-length complement, wherein the variant has chymotrypsin-like activity and has an amino acid sequence that has at least 90% identity to the amino acid sequence of the microbial trypsin. 
     
     
         2 . The variant of  claim 1 , wherein the substitution at position 144 is Thr. 
     
     
         3 . The variant of  claim 1 , wherein the substitution at position 193 is Ala. 
     
     
         4 . The variant of  claim 1 , wherein the substitution at position 198 is Ser. 
     
     
         5 . The variant of  claim 1 , wherein the substitution at position 201 is Met. 
     
     
         6 . The variant of  claim 1 , wherein the substitution at position 218 is Ile. 
     
     
         7 . The variant of  claim 1 , wherein the substitution at position 223 is Ser. 
     
     
         8 . The variant of  claim 1 , wherein the substitution at position 227 is Ser. 
     
     
         9 . The variant of  claim 1 , wherein the substitution at position 228 is Thr. 
     
     
         10 . The variant of  claim 1 , wherein the substitution at position 229 is Ser. 
     
     
         11 . The variant of  claim 1 , wherein the substitution at position 230 is Thr. 
     
     
         12 . The variant of  claim 1 , wherein the substitution at position 231 is Pro. 
     
     
         13 . The variant of  claim 1 , wherein the deletion at position 192 is Val. 
     
     
         14 . The variant of  claim 1 , wherein the deletion at position 197 is Lys. 
     
     
         15 . The variant of  claim 1 , wherein the deletion at position 226 is Ala. 
     
     
         16 . The variant of  claim 1 , wherein the insertion between positions 224 and 225 is Thr. 
     
     
         17 . The variant of  claim 1 , which comprises one or more substitutions selected from the group consisting of V144T, S193A, D198S, Q201M, A218I, N223S, R227S, P228T, N229S, Y230T, and S231 P; one or more deletions selected from the group consisting of V192*, K197*, and A226*; and the insertion G224GT of amino acids 25 to 248 of SEQ ID NO: 2. 
     
     
         18 . The variant of  claim 1 , wherein the microbial trypsin has an amino acid sequence which has at least 90%, 95%, 97%, or 100% identity to amino acids 25 to 248 of SEQ ID NO: 2. 
     
     
         19 . The variant of  claim 1 , wherein the microbial trypsin is encoded by a nucleotide sequence which hybridizes under at least high or very high stringency conditions with nucleotides 202 to 801 of SEQ ID NO: 1. 
     
     
         20 . The variant of  claim 1 , which has an amino acid sequence with at least 90%, 95%, 97%, or 100% identity to the amino acid sequence of the microbial trypsin.