Modified enzymes and methods for making same

Abstract

A cloned subtilsin gene has been modified at specific sites to cause amino acid substitutions at certain spots in the enzyme. The modified enzyme, preferably produced by Bacillus, is useful in combination with detergents.

We claim: .[.1. A subtilisin enzyme having a different amino acid at a site +32, +155, +104, +222, +166, +64, +33, +169, +189, +217, or +157 than subtilisin naturally produced by Bacillus amyloliquefaciens..]. .[.2. A composition comprising an enzyme according to claim 1 in combination with a detergent..]. .[.3. A composition according to claim 2 wherein the detergent is a linear alkyl benzene sulfonate, alkyl benzene sulfate, 
     
        sulfated linear alcohol, or ethoxylated linear alcohol..]. .Iadd.4.  A substantially pure modified subtilisin substituted at the residue position equivalent to asp+32 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.5. A substantially pure modified subtilisin substituted at the residue position equivalent to asn+155 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B, with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.6. A substantially pure modified subtilisin substituted at the residue position equivalent to tyr+104 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen amino acids shown in FIG. 17. .Iaddend. .Iadd.7. A substantially pure modified subtilisin substituted at the residue position equivalent to met+222 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen amino acids as shown in FIG. 17. .Iaddend. .Iadd.8. A substantially pure modified subtilisin substituted at the residue position equivalent to gly+166 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.9. A substantially pure modified subtilisin substituted at the residue position equivalent to his+64 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 
     
     
        17. .Iaddend. .Iadd.10.  A substantially pure modified subtilisin substituted at the residue position equivalent to ser+33 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids as shown in FIG. 17. .Iaddend. .Iadd.11. A substantially pure modified subtilisin substituted at the residue position equivalent to gly+169 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.12. A substantially pure modified subtilisin substituted at the residue position equivalent to phe+189 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.13. A substantially pure modified subtilisin substituted at the residue position equivalent to tyr+217 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.14. A substantially pure modified subtilisin substituted at the residue position equivalent to glu+156 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.15. A substantially pure modified subtilisin substituted at the position equivalent to ala+152 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of the other nineteen naturally occurring amino acids shown in FIG. 17. .Iaddend. .Iadd.16. A modified subtilisin according to claim 7 wherein said subtilisin has an improved pH activity profile when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at the residue position equivalent to met+222. .Iaddend. .Iadd.17. A modified subtilisin according to claim 8 wherein said subtilisin has improved substrate specificity when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at the residue position equivalent to gly+166. .Iaddend. .Iadd.18. A modified subtilisin according to claim 7 wherein said subtilisin has improved oxidative stability when compared to said subtilisin having the amino acid naturally occurring in said subtilisin at the residue position equivalent to met+222. .Iaddend. .Iadd.19. A modified subtilisin according to claim 18 wherein the amino acid substituted at the residue position equivalent to met+222 is selected from the group consisting of ala, ser or cys. .Iaddend. .Iadd.20. A substantially pure modified subtilisin having improved oxidative stability wherein the stability is effected by deleting one or more methionine, tryptophan, cysteine or lysine residue in said subtilisin and substituting another amino acid other than one of methionine, tryptophan, cysteine or lysine, for said deleted amino acid 
     
     
        residue. .Iaddend. .Iadd.21.  A substantially pure modified subtilisin resulting from the expression of DNA which DNA encodes for the amino acid sequence of subtilisin substituted at the residue position equivalent to asp+32, asn+155, tyr+104, met+222, gly+166, his+64, ser+33, gly+169, phe+189, tyr+217, glu+156, or ala+152 of the Bacillus amyloliquefaciens subtilisin shown in FIG. 1B, wherein the substituted amino acid is one of the other nineteen naturally occurring amino acids shown in FIG. 17, and wherein said DNA is obtained by direct recombinant mutagenesis to a precursor DNA. .Iaddend. .Iadd.22. A composition comprising an enzyme according to any one of the claims 4-15 in combination with a detergent. .Iaddend. .Iadd.23. A composition according to claim 22 wherein the detergent is a linear alkyl benzene sulfonate, kyl benzene sulfate, sulfated linear alcohol, or ethoxylated linear alcohol. .Iaddend.