Multiply mutated subtilisins

#The present invention provides a novel class of thermally stable and pH stable subtilisin analogs with improved oxidation stability and superior performance in cleaning soiled fabrics when added to a standard detergent formulation, and to a method for preparing such analogs. In particular, the present invention relates to a class of subtilisin analogs having a modified calcium binding site providing improved calcium binding capacity, and a deletion and/or replacement of either residue of Asn-Gly sequences present in the subtilisin and a modification of one or more methionine residues to improve oxidation stability. The present invention further relates to detergent compositions containing such subtilisins and to the use of such subtilisins and compositions in cleaning applications.

#The term subtilisin designates a group of extracellular alkaline serine proteases produced by various species of Bacilli. These enzymes are also referred to as Bacillus serine proteases, Bacillus subtilisins or bacterial alkaline proteases.

#Bacillus subtilisin molecules are composed of a single polypeptide chain of either 274 residues (for subtilisin type Carlsberg produced by Bacillus licheniformis and for the subtilisin produced by Bacillus subtilis strain DY) or 275 residues (for subtilisin type BPN' produced by Bacillus amyloliquefaciens, the aprA gene product of Bacillus subtilis, and the subtilisin of Bacillus mesentericus and the subtilisin of Bacillus subtilis var. amylosacchariticus). When comparing amino acid sequences of subtilisin from different strains of Bacillus herein, the sequence of subtilisin BPN' is used as a standard. For example, based on an alignment of sequences that gives the highest degree of homology between subtilisin Carlsberg and subtilisin BPN' the serine at the active site of the former is referred to as serine 221, even though it is located at position 220 of the amino acid sequence. On the same basis, position 220 of the amino acid sequence of subtilisin Carlsberg may be said to "correspond" to position 221 of subtilisin BPN'. See e.g., Nedkov et al., Hoppe-Seyler's Z. Physiol. Chem., 364, 1537-1540 (1983).

#FIG. 1 schematically illustrates the cyclization of Asn-Gly residues, such as those found at positions 218 and 219 of subtilisin as set forth in Table 1, to form anhydroaspartylglycine and also depicts base-catalyzed hydrolysis thereof;

#FIG. 2 is a partial restriction map of an aprA gene-containing an EcoRI-KpnI gene fragment of Bacillus subtilis (B. subtilis) strain QB127 and includes a partial restriction map of the aprA gene and flanking sequences;

#FIG. 3 is a partial restriction map of a plasmid pAMB11;

#The term "subtilisin" as employed herein, refers to a mature, secreted form of the enzyme which lacks leader sequences cleaved from the mature enzyme prior to or at secretion. Subtilisins that may be modified in accordance with the present invention include, but are not limited to, naturally occurring subtilisins represented by the amino acid sequence of subtilisin Carlsberg, subtilisin BPN' the aprA gene product of Bacillus subtilis, subtilisin DY and the subtilisin of Bacillus mesentericus, and the subtilisin of B. subtilis var. Amylosacchariticus. The amino acid sequence for subtilisin Carlsberg is described by Smith, et al., J. Biol. Chem., 243, 2184-2191 (1968). The amino acid sequence for subtilisin BPN' is described by Markland, et al., J. Biol. Chem., 242, 5198-5211 (1967). The amino acid sequence for subtilisin DY is described by Nedlov, et al., Hoppe-Seyler's Z. Physiol. Chem., 364, 1537-1540 (1983). The amino acid sequence for the subtilisin of Bacillus mesentericus is described by Svedsen, et al., FEBS Letters, 196, 220-232 (1986 ). The amino acid sequence for the subtilisin of Bacillus subtilis var. amylosacchariticus is described by Yoshimoto, et al., J. Biochem., 103, 1060-1065 (1988). The amino acid sequence of the aprA gene product of Bacillus subtilis is described by Stahl, et al., J. Bacteriol., 158, 411-418 (1984). The amino acid sequence of such subtilisins are incorporated by reference herein. Such subtilisins are characterized as having calcium binding sites necessary to stabilize the molecule.

#In accordance with the present invention, a class of subtilisin analogs are provided which possess improved capacity to bind to calcium. Calcium has been used to stabilize subtilisin in powders and liquid detergent, especially in applications requiring higher temperatures. The present invention relates to the modification of the calcium binding site of the subtilisin molecule to increase calcium binding. As used herein the term "modification of the calcium binding site" refers to replacement of one or more amino acids in the region of a calcium binding site present in the amino acid sequence of subtilisin with a negatively charged amino acid thereby enabling the resulting subtilisin analog to have an additional negative charge. It has been found that one calcium binding site in a subtilisin involves the following amino acids: Gln2, Asp.sup.41, Leu.sup.75, Asn.sup.76, Asn.sup.77, Ser.sup.78, Ile.sup.79, Gly.sup.80, Val.sup.81, Thr.sup.208 and Tyr.sup.214 relative to the amino acid sequence set forth in Table 1. The present invention preferably involves replacement of one or more of the amino acids present in the calcium binding site with a "negatively charged" amino acid such as Asp and Glu, and more preferably Asp. It should be noted that although Asp.sup.41 in the calcium binding site is a negatively charged amino acid, one embodiment of the present invention involves changing Asp.sup.41 to Glu.sup.41. The other embodiments relate to changes other than to Asp.sup.41.

#One preferred embodiment of the present invention involves a subtilisin analog wherein Asn.sup.76 is converted to Asp.sup.76. Another embodiment involves conversion of the Ile.sup.79 to Asp.sup.79. Another embodiment involves the above preferred modifications to the calcium binding site and substitutions of Asn.sup.109 and Asn.sup.218 to Ser.sup.109 and Ser.sup.218, thus eliminating two unstable Asn-Gly sequences. A more preferred embodiment of the present invention involves the above preferred modifications to the calcium binding site and Asn-Gly sequences in combination with changes of methionine 222 to alanine. Another preferred embodiment involves the above preferred modifications to the calcium binding site and Asn-Gly sequences and Met-222 in combination with amino acid modifications that increase the specificity of subtilisin to an azocasein substrate or a sAAPFpN substrate i.e., Methionine 124 to Leucine or Alanine and/or Isoleucine 31 to Leucine, and/or Serine 33 to Threonine, and/or Serine 62 to Asparagine, and/or Serine 63 to Glycine, and/or Tyrosine 217 to Leucine and/or Arginine 247 to Leucine or Methionine.