Methods for obtaining microbial trypsin mutants having chymotrypsin activity

1. 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.

2. The method of claim 1 , wherein the variant is encoded by the nucleotide sequence contained in pEJG66.1XLGOLD, which is contained in E. coli NRRL B-30627.

3. The method of claim 1 , wherein the variant is in a form of a precursor comprising amino acids 1 to 24 of SEQ ID NO: 2 as a prepro region, or a portion thereof, linked in translation reading frame with the amino terminus of the variant.

4. The method of claim 1 , which comprises the substitutions V144T+S193A+D198S+Q201M+A218I+N223S+R227S+P228T+N229S+Y230T+5231 P, the deletions V192*+K197*+A226*, and the insertion G224GT of amino acids 25 to 248 of SEQ ID NO: 2.

5. The method of claim 1 , wherein the trypsin polypeptide has an amino acid sequence which has at least 90% identity to amino acids 25 to 248 of SEQ ID NO. 2.

6. The method of claim 5 , wherein the trypsin polypeptide has an amino acid sequence which has at least 95% identity to amino acids 25 to 248 of SEQ ID NO: 2.

7. The method of claim 6 , wherein the trypsin polypeptide has an amino acid sequence which has at least 97% identity to amino acids 25 to 248 of SEQ ID NO: 2.

8. The method of claim 1 , wherein the trypsin polypeptide has the amino acid sequence of amino acids 25 to 248 of SEQ ID NO: 2.

9. The method of claim 1 , wherein the trypsin polypeptide is encoded by a nucleotide sequence which hybridizes under 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.

10. The method of claim 9 , wherein the trypsin polypeptide is encoded by a nucleotide sequence which hybridizes under high stringency conditions with nucleotides 202 to 801 of SEQ ID NO: 1 or the complete complementary strand thereto, wherein 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 50% formamide followed by washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

11. The method of claim 10 , wherein the trypsin polypeptide is encoded by a nucleotide sequence which hybridizes under very high stringency conditions with nucleotides 202 to 801 of SEQ ID NO: 1 or the complete complementary strand thereto, wherein very 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 50% formamide followed by washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

12. The method of claim 1 , wherein the trypsin polypeptide is a wild-type trypsin polypeptide.

13. The method of claim 1 , wherein the variant comprises amino acids 25 to 246 of SEQ ID NO: 4.

14. The method of claim 1 , wherein the variant has at least 90% identity to amino acids 25 to 248 of SEQ ID NO: 2.