BACKGROUND OF THE INVENTION
 Serine proteases are a subgroup of carbonyl hydrolases. They comprise a
 diverse class of enzymes having a wide range of specificities and
 biological functions. Stroud, R. Sci. Amer., 131:74-88. Despite their
 functional diversity, the catalytic machinery of serine proteases has been
 approached by at least two genetically distinct families of enzymes: 1)
 the subtilisins and 2) the mammalian chymotrypsin-related and homologous
 bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These
 two families of serine proteases show remarkably similar mechanisms of
 catalysis. Kraut, J. (1977), Annu. Rev. Biochem., 46:331-358. Furthermore,
 although the primary structure is unrelated, the tertiary structure of
 these two enzyme families bring together a conserved catalytic triad of
 amino acids consisting of serine, histidine and aspartate.
 Subtilisins are serine proteases (approx. MW 27,500) which are secreted in
 large amounts from a wide variety of Bacillus species and other
 microorganisms. The protein sequence of subtilisin has been determined
 from at least nine different species of Bacillus. Markland, F. S., et al.
 (1983), Hoppe-Seyler's Z. Physiol. Chem., 364:1537-1540. The
 three-dimensional crystallographic structure of subtilisins from Bacillus
 amyloliquefaciens, Bacillus licheniforimis and several natural variants of
 B.lentus have been reported. These studies indicate that although
 subtilisin is genetically unrelated to the mammalian serine proteases, it
 has a similar active site structure. The x-ray crystal structures of
 subtilisin containing covalently bound peptide inhibitors (Robertus, J.
 D., et al. (1972), Biochemistry, 11:2439-2449) or product complexes
 (Robertus, J. D., et al. (1976), J. Biol. Chem., 251:1097-1103) have also
 provided information regarding the active site and putative substrate
 binding cleft of subtilisin. In addition, a large number of kinetic and
 chemical modification studies have been reported for subtilisin; Svendsen,
 B. (1976), Carlsberg Res. Commun., 41:237-291; Markland, F. S. Id.) as
 well as at least one report wherein the side chain of methionine at
 residue 222 of subtilisin was converted by hydrogen peroxide to
 methionine-sulfoxide (Stauffer, D. C., et al. (1965), J. Biol. Chem.,
 244:5333-5338) and extensive site-specific mutagenesis has been carried
 out (Wells and Estell (1988) TIBS 13:291-297)
 SUMMARY OF THE INVENTION
 It is an object herein to provide protease variants containing a
 substitution of an amino acid at a residue position corresponding to
 position 103 of Bacillus amyloliquefaciens subtilisin and substituting one
 or more amino acids at residue positions selected from the group
 consisting of residue positions corresponding to positions 1, 3, 4, 8, 10,
 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38, 42, 43, 48, 55,
 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97, 98, 99, 101,
 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123, 126, 128, 130,
 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159, 160, 166, 167, 170,
 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198, 203, 204, 205,
 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222, 224, 227, 228,
 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247, 248, 249, 251,
 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 268, 269,
 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens subtilisin;
 wherein when a substitution at a position corresponding to residue
 position 103 is combined with a substitution at a position corresponding
 to residue position 76, there is also a substitution at one or more
 residue positions other than residue positions corresponding to positions
 27, 99, 101, 104, 107, 109, 123, 128, 166, 204, 206, 210, 216, 217, 218,
 222, 260, 265, or 274 of Bacillus amyloliquefaciens subtilisin.
 While any combination of the above listed amino acid substitutions may be
 employed, the preferred protease variant enzymes useful for the present
 invention comprise the substitution of amino acid residues in the
 following combinations of positions. All of the residue positions
 correspond to positions of Bacillus amyloliquefaciens subtilisin:
 (1) a protease variant including substitutions of the amino acid residues
 at position 103 and at one or more of the following positions 236 and 245;
 (2) a protease variant including substitutions of the amino acid residues
 at positions 103 and 236 and at one or more of the following positions 1,
 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 183,
 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 248, 252, 257, 260,
 270 and 275;
 (3) a protease variant including substitutions of the amino acid residues
 at positions 103 and 245 and at one or more of the following positions 1,
 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131, 159, 170,
 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 222, 230, 232, 248, 252,
 257, 260, 261, 270 and 275; or
 (4) a protease variant including substitutions of the amino acid residues
 at positions 103, 236 and 245 and at one or more of the following
 positions 1, 9, 12, 61, 62, 68, 76, 97, 98, 101, 102, 104, 109, 130, 131,
 159, 183, 185, 205, 209, 210, 211, 212, 213, 215, 217, 230, 232, 243, 248,
 252, 257, 260, 270 and 275.
 More preferred protease variants are substitution sets selected from the
 group consisting of residue positions corresponding to positions in Table
 1 of Bacillus amyloliquefaciens subtilisin:

DETAILED DESCRIPTION OF THE INVENTION
 Proteases are carbonyl hydrolases which generally act to cleave peptide
 bonds of proteins or peptides. As used herein, "protease" means a
 naturally-occurring protease or a recombinant protease.
 Naturally-occurring proteases include .alpha.-aminoacylpeptide hydrolase,
 peptidylamino acid hydrolase, acylamino hydrolase, serine
 carboxypeptidase, metallocarboxypeptidase, thiol proteinase,
 carboxylproteinase and metalloproteinase. Serine, metallo, thiol and acid
 proteases are included, as well as endo and exo-proteases,
 The present invention includes protease enzymes which are non-naturally
 occurring carbonyl hydrolase variants (protease variants) having a
 different proteolytic activity, stability, substrate specificity, pH
 profile and/or performance characteristic as compared to the precursor
 carbonyl hydrolase from which the amino acid sequence of the variant is
 derived. Specifically, such protease variants have an amino acid sequence
 not found in nature, which is derived by substitution of a plurality of
 amino acid residues of a precursor protease with different amino acids.
 The precursor protease may be a naturally-occurring protease or a
 recombinant protease.
 The protease variants useful herein encompass the substitution of any of
 the nineteen naturally occurring L-amino acids at the designated amino
 acid residue positions. Such substitutions can be made in any precursor
 subtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout this
 application reference is made to various amino acids by way of common
 one--and three-letter codes. Such codes are identified in Dale, M. W.
 (1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd., Appendix
 B.
 The protease variants useful herein are preferably derived from a Bacillus
 subtilisin. More preferably, the protease variants are derived from
 Bacillus lentus subtilisin and/or subtilisin 309.
 Subtilisins are bacterial or fungal proteases which generally act to cleave
 peptide bonds of proteins or peptides. As used herein, "subtilisin" means
 a naturally-occurring subtilisin or a recombinant subtilisin. A series of
 naturally-occurring subtilisins is known to be produced and often secreted
 by various microbial species. Amino acid sequences of the members of this
 series are not entirely homologous. However, the subtilisins in this
 series exhibit the same or similar type of proteolytic activity. This
 class of serine proteases shares a common amino acid sequence defining a
 catalytic triad which distinguishes them from the chymotrypsin related
 class of serine proteases. The subtilisins and chymotrypsin related serine
 proteases both have a catalytic triad comprising aspartate, histidine and
 serine. In the subtilisin related proteases the relative order of these
 amino acids, reading from the amino to carboxy terminus, is
 aspartate-histidine-serine. In the chymotrypsin related proteases, the
 relative order, however, is histidine-aspartate-serine. Thus, subtilisin
 herein refers to a serine protease having the catalytic triad of
 subtilisin related proteases. Examples include but are not limited to the
 subtilisins identified in FIG. 3 herein. Generally and for purposes of the
 present invention, numbering of the amino acids in proteases corresponds
 to the numbers assigned to the mature Bacillus amyloliquefaciens
 subtilisin sequence presented in FIG. 1.
 "Recombinant subtilisin" or "recombinant protease" refer to a subtilisin or
 protease in which the DNA sequence encoding the subtilisin or protease is
 modified to produce a variant (or mutant) DNA sequence which encodes the
 substitution, deletion or insertion of one or more amino acids in the
 naturally-occurring amino acid sequence. Suitable methods to produce such
 modification, and which may be combined with those disclosed herein,
 include those disclosed in U.S. Pat. Nos. RE 34,606, 5,204,015 and
 5,185,258, 5,700,676, 5,801,038, and 5,763,257.
 "Non-human subtilisins" and the DNA encoding them may be obtained from many
 procaryotic and eucaryotic organisms. Suitable examples of procaryotic
 organisms include gram negative organisms such as E. coli or Pseudomonas
 and gram positive bacteria such as Micrococcus or Bacillus. Examples of
 eucaryotic organisms from which subtilisin and their genes may be obtained
 include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus
 sp.
 A "protease variant" has an amino acid sequence which is derived from the
 amino acid sequence of a "precursor protease". The precursor proteases
 include naturally-occurring proteases and recombinant proteases. The amino
 acid sequence of the protease variant is "derived" from the precursor
 protease amino acid sequence by the substitution, deletion or insertion of
 one or more amino acids of the precursor amino acid sequence. Such
 modification is of the "precursor DNA sequence" which encodes the amino
 acid sequence of the precursor protease rather than manipulation of the
 precursor protease enzyme per se. Suitable methods for such manipulation
 of the precursor DNA sequence include methods disclosed herein, as well as
 methods known to those skilled in the art (see, for example, EP 0 328299,
 WO89/06279 and the US patents and applications already referenced herein).
 Specific substitutions corresponding to position 103 in combination with
 one or more of the following substitutions corresponding to positions 1,
 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27, 33, 37, 38 42,
 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79, 86, 87, 89, 97,
 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117, 119, 121, 123,
 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147, 158, 159 160,
 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188, 192, 194, 198,
 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 222,
 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244, 245, 246, 247,
 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263,
 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus amyloliquefaciens
 subtilisin are identified herein.
 Preferred variants are those having combinations of substitutions at
 residue positions corresponding to positions of Bacillus amyloliquefaciens
 subtilisin in Table 1. More preferred variants are those having
 combinations of substitutions at residue positions corresponding to
 positions of Bacillus amyloliquefaciens subtilisin in Table 3.
 Further preferred variants are those having combinations of substitutions
 at residue positions corresponding to positions of Bacillus
 amyloliquefaciens subtilisin in Table 2.
 TABLE 2
 76 103 104 222 245
 68 103 104 159 232 236 245 248 252
 68 103 104 159 232 236 245
 68 103 104 140 159 232 236 245 252
 43 68 103 104 159 232 236 245 252
 43 68 103 104 159 232 236 245
 12 76 103 104 130 222 245 261
 76 103 104 130 222 245
 68 103 104 159 232 236 245 257
 68 76 103 104 159 210 232 236 245
 68 103 104 159 224 232 236 245 257
 76 103 104 159 232 236 245 257
 68 76 103 104 159 211 232 236 245
 12 68 76 103 104 159 214 232 236 245
 68 76 103 104 159 215 232 236 245
 12 68 76 103 104 159 232 236 245
 20 68 76 103 104 159 232 236 245 259
 68 76 87 103 104 159 232 236 245 260
 68 76 103 104 159 232 236 245 261
 12 48 68 76 103 104 159 232 236 245
 76 103 104 159 192 232 236 245
 76 103 104 147 159 232 236 245 248 251
 12 68 76 103 104 159 232 236 245 272
 68 76 103 104 159 183 206 232 236 245
 68 76 103 104 159 232 236 245 256
 68 76 103 104 159 206 232 236 245
 27 68 76 103 104 159 232 236 245
 68 103 104 159 212 232 236 245 248 252
 103 104 159 232 236 245 248 252
 68 103 104 159 209 232 236 245 248 252
 68 103 104 109 159 232 236 245 248 252
 20 68 103 104 159 232 236 245 248 252
 68 103 104 159 209 232 236 245 248 252
 68 103 104 159 210 232 236 245 248 252
 68 103 104 159 212 232 236 245 248 252
 68 103 104 159 213 232 236 245 248 252
 68 103 104 213 232 236 245 248 252
 68 103 104 159 215 232 236 245 248 252
 68 103 104 159 216 232 236 245 248 252
 20 68 103 104 159 232 236 245 248 252
 68 103 104 159 232 236 245 248 252 255
 68 103 104 159 232 236 245 248 252 256
 68 103 104 159 232 236 245 248 252 260
 68 103 104 159 228 232 236 245 248 252
 68 76 89 103 104 159 210 213 232 236 245 260
 68 103 104 159 218 232 236 245 248 252
 These amino acid position numbers refer to those assigned to the mature
 Bacillus amyloliquefaciens subtilisin sequence presented in FIG. 1. The
 invention, however, is not limited to the mutation of this particular
 subtilisin but extends to precursor proteases containing amino acid
 residues at positions which are "equivalent" to the particular identified
 residues in Bacillus amyloliquefaciens subtilisin. In a preferred
 embodiment of the present invention, the precursor protease is Bacillus
 lentus subtilisin and the substitutions are made at the equivalent amino
 acid residue positions in B. lentus corresponding to those listed above.
 A residue (amino acid) position of a precursor protease is equivalent to a
 residue of Bacillus amyloliquefaciens subtilisin if it is either
 homologous (i.e., corresponding in position in either primary or tertiary
 structure) or analogous to a specific residue or portion of that residue
 in Bacillus amyloliquefaciens subtilisin (i.e., having the same or similar
 functional capacity to combine, react, or interact chemically).
 In order to establish homology to primary structure, the amino acid
 sequence of a precursor protease is directly compared to the Bacillus
 amyloliquefaciens subtilisin primary sequence and particularly to a set of
 residues known to be invariant in subtilisins for which sequence is known.
 For example, FIG. 2 herein shows the conserved residues as between B.
 amyloliquefaciens subtilisin and B. lentus subtilisin. After aligning the
 conserved residues, allowing for necessary insertions and deletions in
 order to maintain alignment (i.e., avoiding the elimination of conserved
 residues through arbitrary deletion and insertion), the residues
 equivalent to particular amino acids in the primary sequence of Bacillus
 amyloliquefaciens subtilisin are defined. Alignment of conserved residues
 preferably should conserve 100% of such residues. However, alignment of
 greater than 75% or as little as 50% of conserved residues is also
 adequate to define equivalent residues. Conservation of the catalytic
 triad, Asp32/His64/Ser221 should be maintained. Siezen et al. (1991)
 Protein Eng. 4(7):719-737 shows the alignment of a large number of serine
 proteases. Siezen et al. refer to the grouping as subtilases or
 subtilisin-like serine proteases.
 For example, in FIG. 3, the amino acid sequence of subtilisin from Bacillus
 amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis
 (carlsbergensis) and Bacillus lentus are aligned to provide the maximum
 amount of homology between amino acid sequences. A comparison of these
 sequences shows that there are a number of conserved residues contained in
 each sequence. These conserved residues (as between BPN' and B. lentus)
 are identified in FIG. 2.
 These conserved residues, thus, may be used to define the corresponding
 equivalent amino acid residues of Bacillus amyloliquefaciens subtilisin in
 other subtilisins such as subtilisin from Bacillus lentus (PCT Publication
 No. WO89/06279 published Jul. 13, 1989), the preferred protease precursor
 enzyme herein, or the subtilisin referred to as PB92 (EP 0 328 299), which
 is highly homologous to the preferred Bacillus lentus subtilisin. The
 amino acid sequences of certain of these subtilisins are aligned in FIGS.
 3A and 3B with the sequence of Bacillus amyloliquefaciens subtilisin to
 produce the maximum homology of conserved residues. As can be seen, there
 are a number of deletions in the sequence of Bacillus lentus as compared
 to Bacillus amyloliquefaciens subtilisin. Thus, for example, the
 equivalent amino acid for Val165 in Bacillus amyloliquefaciens subtilisin
 in the other subtilisins is isoleucine for B. lentus and B. licheniformis.
 "Equivalent residues" may also be defined by determining homology at the
 level of tertiary structure for a precursor protease whose tertiary
 structure has been determined by x-ray crystallography. Equivalent
 residues are defined as those for which the atomic coordinates of two or
 more of the main chain atoms of a particular amino acid residue of the
 precursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CA
 on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm after
 alignment. Alignment is achieved after the best model has been oriented
 and positioned to give the maximum overlap of atomic coordinates of
 non-hydrogen protein atoms of the protease in question to the Bacillus
 amyloliquefaciens subtilisin. The best model is the crystallographic model
 giving the lowest R factor for experimental diffraction data at the
 highest resolution available.
 ##EQU1##
 Equivalent residues which are functionally analogous to a specific residue
 of Bacillus amyloliquefaciens subtilisin are defined as those amino acids
 of the precursor protease which may adopt a conformation such that they
 either alter, modify or contribute to protein structure, substrate binding
 or catalysis in a manner defined and attributed to a specific residue of
 the Bacillus amyloliquefaciens subtilisin. Further, they are those
 residues of the precursor protease (for which a tertiary structure has
 been obtained by x-ray crystallography) which occupy an analogous position
 to the extent that, although the main chain atoms of the given residue may
 not satisfy the criteria of equivalence on the basis of occupying a
 homologous position, the atomic coordinates of at least two of the side
 chain atoms of the residue lie with 0.13 nm of the corresponding side
 chain atoms of Bacillus amyloliquefaciens subtilisin. The coordinates of
 the three dimensional structure of Bacillus amyloliquefaciens subtilisin
 are set forth in EPO Publication No. 0 251 446 (equivalent to U.S. Pat.
 No. 5,182,204, the disclosure of which is incorporated herein by
 reference) and can be used as outlined above to determine equivalent
 residues on the level of tertiary structure.
 Some of the residues identified for substitution are conserved residues
 whereas others are not. In the case of residues which are not conserved,
 the substitution of one or more amino acids is limited to substitutions
 which produce a variant which has an amino acid sequence that does not
 correspond to one found in nature. In the case of conserved residues, such
 substitutions should not result in a naturally-occurring sequence. The
 protease variants of the present invention include the mature forms of
 protease variants, as well as the pro- and prepro-forms of such protease
 variants. The prepro-forms are the preferred construction since this
 facilitates the expression, secretion and maturation of the protease
 variants.
 "Prosequence" refers to a sequence of amino acids bound to the N-terminal
 portion of the mature form of a protease which when removed results in the
 appearance of the "mature" form of the protease. Many proteolytic enzymes
 are found in nature as translational proenzyme products and, in the
 absence of post-translational processing, are expressed in this fashion. A
 preferred prosequence for producing protease variants is the putative
 prosequence of Bacillus amyloliquefaciens subtilisin, although other
 protease prosequences may be used.
 A "signal sequence" or "presequence" refers to any sequence of amino acids
 bound to the N-terminal portion of a protease or to the N-terminal portion
 of a proprotease which may participate in the secretion of the mature or
 pro forms of the protease. This definition of signal sequence is a
 functional one, meant to include all those amino acid sequences encoded by
 the N-terminal portion of the protease gene which participate in the
 effectuation of the secretion of protease under native conditions. The
 present invention utilizes such sequences to effect the secretion of the
 protease variants as defined herein. One possible signal sequence
 comprises the first seven amino acid residues of the signal sequence from
 Bacillus subtilis subtilisin fused to the remainder of the signal sequence
 of the subtilisin from Bacillus lentus (ATCC 21536).
 A "prepro" form of a protease variant consists of the mature form of the
 protease having a prosequence operably linked to the amino terminus of the
 protease and a "pre" or "signal" sequence operably linked to the amino
 terminus of the prosequence.
 "Expression vector" refers to a DNA construct containing a DNA sequence
 which is operably linked to a suitable control sequence capable of
 effecting the expression of said DNA in a suitable host Such control
 sequences include a promoter to effect transcription, an optional operator
 sequence to control such transcription, a sequence encoding suitable mRNA
 ribosome binding sites and sequences which control termination of
 transcription and translation. The vector may be a plasmid, a phage
 particle, or simply a potential genomic insert. Once transformed into a
 suitable host, the vector may replicate and function independently of the
 host genome, or may, in some instances, integrate into the genome itself.
 In the present specification, "plasmid" and "vector" are sometimes used
 interchangeably as the plasmid is the most commonly used form of vector at
 present. However, the invention is intended to include such other forms of
 expression vectors which serve equivalent functions and which are, or
 become, known in the art.
 The "host cells" used in the present invention generally are procaryotic or
 eucaryotic hosts which preferably have been manipulated by the methods
 disclosed in U.S. Pat. No. RE 34,606 to render them incapable of secreting
 enzymatically active endoprotease. A preferred host cell for expressing
 protease is the Bacillus strain BG2036 which is deficient in enzymatically
 active neutral protease and alkaline protease (subtilisin). The
 construction of strain BG2036 is described in detail in U.S. Pat. No.
 5,264,366. Other host cells for expressing protease include Bacillus
 subtilis I168 (also described in U.S. Pat. No. RE 34,606 and U.S. Pat. No.
 5,264,366, the disclosure of which are incorporated herein by reference),
 as well as any suitable Bacillus strain such as B. licheniformis, B.
 lentus, etc.
 Host cells are transformed or transfected with vectors constructed using
 recombinant DNA techniques. Such transformed host cells are capable of
 either replicating vectors encoding the protease variants or expressing
 the desired protease variant, In the case of vectors which encode the pre-
 or prepro-form of the protease variant, such variants when expressed, are
 typically secreted from the host cell into the host cell medium.
 "Operably linked,"when describing the relationship between two DNA regions,
 simply means that they are functionally related to each other. For
 example, a presequence is operably linked to a peptide if it functions as
 a signal sequence, participating in the secretion of the mature form of
 the protein most probably involving cleavage of the signal sequence. A
 promoter is operably linked to a coding sequence if it controls the
 transcription of the sequence; a ribosome binding site is operably linked
 to a coding sequence if it is positioned so as to permit translation.
 The genes encoding the naturally-occurring precursor protease may be
 obtained in accord with the general methods known to those skilled in the
 art. The methods generally comprise synthesizing labeled probes having
 putative sequences encoding regions of the protease of interest, preparing
 genomic libraries from organisms expressing the protease, and screening
 the libraries for the gene of interest by hybridization to the probes.
 Positively hybridizing clones are then mapped and sequenced.
 The cloned protease is then used to transform a host cell in order to
 express the protease. The protease gene is then ligated into a high copy
 number plasmid. This plasmid replicates in hosts in the sense that it
 contains the well-known elements necessary for plasmid replication: a
 promoter operably linked to the gene in question (which may be supplied as
 the gene's own homologous promoter if it is recognized, i.e., transcribed,
 by the host), a transcription termination and polyadenylation region
 (necessary for stability of the mRNA transcribed by the host from the
 protease gene in certain eucaryotic host cells) which is exogenous or is
 supplied by the endogenous terminator region of the protease gene and,
 desirably, a selection gene such as an antibiotic resistance gene that
 enables continuous cultural maintenance of plasmid-infected host cells by
 growth in antibiotic-containing media. High copy number plasmids also
 contain an origin of replication for the host, thereby enabling large
 numbers of plasmids to be generated in the cytoplasm without chromosomal
 limitations. However, it is within the scope herein to integrate multiple
 copies of the protease gene into host genome. This is facilitated by
 procaryotic and eucaryotic organisms which are particularly susceptible to
 homologous recombination.
 The gene can be a natural B. lentus gene. Alternatively, a synthetic gene
 encoding a naturally-occurring or mutant precursor protease may be
 produced. In such an approach, the DNA and/or amino acid sequence of the
 precursor protease is determined. Multiple, overlapping synthetic
 single-stranded DNA fragments are thereafter synthesized, which upon
 hybridization and ligation produce a synthetic DNA encoding the precursor
 protease. An example of synthetic gene construction is set forth in
 Example 3 of U.S. Pat. No. 5,204,015, the disclosure of which is
 incorporated herein by reference.
 Once the naturally-occurring or synthetic precursor protease gene has been
 cloned, a number of modifications are undertaken to enhance the use of the
 gene beyond synthesis of the naturally-occurring precursor protease. Such
 modifications include the production of recombinant proteases as disclosed
 in U.S. Pat. No. RE 34,606 and EPO Publication No. 0 251 446 and the
 production of protease variants described herein.
 The following cassette mutagenesis method may be used to facilitate the
 construction of the protease variants of the present invention, although
 other methods may be used. First, the naturally-occurring gene encoding
 the protease is obtained and sequenced in whole or in part. Then the
 sequence is scanned for a point at which it is desired to make a mutation
 (deletion, insertion or substitution) of one or more amino acids in the
 encoded enzyme. The sequences flanking this point are evaluated for the
 presence of restriction sites for replacing a short segment of the gene
 with an oligonucleotide pool which when expressed will encode various
 mutants. Such restriction sites are preferably unique sites within the
 protease gene so as to facilitate the replacement of the gene segment.
 However, any convenient restriction site which is not overly redundant in
 the protease gene may be used, provided the gene fragments generated by
 restriction digestion can be reassembled in proper sequence. If
 restriction sites are not present at locations within a convenient
 distance from the selected point (from 10 to 15 nucleotides), such sites
 are generated by substituting nucleotides in the gene in such a fashion
 that neither the reading frame nor the amino acids encoded are changed in
 the final construction. Mutation of the gene in order to change its
 sequence to conform to the desired sequence is accomplished by M13 primer
 extension in accord with generally known methods. The task of locating
 suitable flanking regions and evaluating the needed changes to arrive at
 two convenient restriction site sequences is made routine by the
 redundancy of the genetic code, a restriction enzyme map of the gene and
 the large number of different restriction enzymes. Note that if a
 convenient flanking restriction site is available, the above method need
 be used only in connection with the flanking region which does not contain
 a site.
 Once the naturally-occurring DNA or synthetic DNA is cloned, the
 restriction sites flanking the positions to be mutated are digested with
 the cognate restriction enzymes and a plurality of end
 termini-complementary oligonucleotide cassettes are ligated into the gene.
 The mutagenesis is simplified by this method because all of the
 oligonucleotides can be synthesized so as to have the same restriction
 sites, and no synthetic linkers are necessary to create the restriction
 sites.
 As used herein, proteolytic activity is defined as the rate of hydrolysis
 of peptide bonds per milligram of active enzyme. Many well known
 procedures exist for measuring proteolytic activity (K. M. Kalisz,
 "Microbial Proteinases," Advances in Biochemical
 Engineering/Biotechnology, A. Fiechter ed., 1988). In addition to or as an
 alternative to modified proteolytic activity, the variant enzymes of the
 present invention may have other modified properties such as K.sub.m,
 k.sub.cat, k.sub.cat /K.sub.m ratio and/or modified substrate specificity
 and/or modified pH activity profile. These enzymes can be tailored for the
 particular substrate which is anticipated to be present, for example, in
 the preparation of peptides or for hydrolytic processes such as laundry
 uses.
 In one aspect of the invention, the objective is to secure a variant
 protease having altered, preferably improved wash performance as compared
 to a precursor protease in at least one detergent formulation and or under
 at least one set of wash conditions.
 There is a variety of wash conditions including varying detergent
 formulations, wash water volume, wash water temperature and length of wash
 time that a protease variant might be exposed to. For example, detergent
 formulations used in different areas have different concentrations of
 their relevant components present in the wash water. For example, a
 European detergent typically has about 4500-5000 ppm of detergent
 components in the wash water while a Japanese detergent typically has
 approximately 667 ppm of detergent components in the wash water. In North
 America, particularly the United States, a detergent typically has about
 975 ppm of detergent components present in the wash water.
 A low detergent concentration system includes detergents where less than
 about 800 ppm of detergent components are present in the wash water.
 Japanese detergents are typically considered low detergent concentration
 system as they have approximately 667 ppm of detergent components present
 in the wash water.
 A medium detergent concentration includes detergents where between about
 800 ppm and about 2000ppm of detergent components are present in the wash
 water. North American detergents are generally considered to be medium
 detergent concentration systems as they have approximately 975 ppm of
 detergent components present in the wash water. Brazil typically has
 approximately 1500 ppm of detergent components present in the wash water.
 A high detergent concentration system includes detergents where greater
 than about 2000 ppm of detergent components are present in the wash water.
 European detergents are generally considered to be high detergent
 concentration systems as they have approximately 4500-5000 ppm of
 detergent components in the wash water.
 Latin American detergents are generally high suds phosphate builder
 detergents and the range of detergents used in Latin America can fall in
 both the medium and high detergent concentrations as they range from 1500
 ppm to 6000 ppm of detergent components in the wash water. As mentioned
 above, Brazil typically has approximately 1500 ppm of detergent components
 present in the wash water. However, other high suds phosphate builder
 detergent geographies, not limited to other Latin American countries, may
 have high detergent concentration systems up to about 6000 ppm of
 detergent components present in the wash water.
 In light of the foregoing, it is evident that concentrations of detergent
 compositions in typical wash solutions throughout the world varies from
 less than about 800 ppm of detergent composition ("low detergent
 concentration geographies"), for example about 667 ppm in Japan, to
 between about 800 ppm to about 2000 ppm ("medium detergent concentration
 geographies" ), for example about 975 ppm in U.S. and about 1500 ppm in
 Brazil, to greater than about 2000 ppm ("high detergent concentration
 geographies"), for example about 4500 ppm to about 5000 ppm in Europe and
 about 6000 ppm in high suds phosphate builder geographies.
 The concentrations of the typical wash solutions are determined
 empirically. For example, in the U.S., a typical washing machine holds a
 volume of about 64.4 L of wash solution. Accordingly, in order to obtain a
 concentration of about 975 ppm of detergent within the wash solution about
 62.79 g of detergent composition must be added to the 64.4 L of wash
 solution. This amount is the typical amount measured into the wash water
 by the consumer using the measuring cup provided with the detergent.
 As a further example, different geographies use different wash
 temperatures. The temperature of the wash water in Japan is typically less
 than that used in Europe.
 Accordingly one aspect of the present invention includes a protease variant
 that shows improved wash performance in at least one set of wash
 conditions.
 In another aspect of the invention, it has been determined that
 substitutions at a position corresponding to 103 in combination with one
 or more substitutions selected from the group consisting of positions
 corresponding 1, 3, 4, 8, 10, 12, 13, 16, 17, 18, 19, 20, 21, 22, 24, 27,
 33, 37, 38, 42, 43, 48, 55, 57, 58, 61, 62, 68, 72, 75, 76, 77, 78, 79,
 86, 87, 89, 97, 98, 99, 101, 102, 104, 106, 107, 109, 111, 114, 116, 117,
 119, 121, 123, 126, 128, 130, 131, 133, 134, 137, 140, 141, 142, 146, 147,
 158, 159, 160, 166, 167, 170, 173, 174, 177, 181, 182, 183, 184, 185, 188,
 192, 194, 198, 203, 204, 205, 206, 209, 210, 211, 212, 213, 214, 215, 216,
 217, 218, 222, 224, 227, 228, 230, 232, 236, 237, 238, 240, 242, 243, 244,
 245, 246, 247, 248, 249, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,
 261, 262, 263, 265, 268, 269, 270, 271, 272, 274 and 275 of Bacillus
 amyloliquefaciens subtilisin are important in improving the wash
 performance of the enzyme.
 These substitutions are preferably made in Bacillus lentus (recombinant or
 native-type) subtilisin, although the substitutions may be made in any
 Bacillus protease.
 Based on the screening results obtained with the variant proteases, the
 noted mutations in Bacillus amyloliquefaciens subtilisin are important to
 the proteolytic activity, performance and/or stability of these enzymes
 and the cleaning or wash performance of such variant enzymes.
 Many of the protease variants of the invention are useful in formulating
 various detergent compositions or personal care formulations such as
 shampoos or lotions. A number of known compounds are suitable surfactants
 useful in compositions comprising the protease mutants of the invention.
 These include nonionic, anionic, cationic, or zwitterionic detergents, as
 disclosed in U.S. Pat. No. 4,404,128 to Barry J. Anderson and U.S. Pat.
 No. 4,261,868 to Jiri Flora, et al. A suitable detergent formulation is
 that described in Example 7 of U.S. Pat. No. 5,204,015 (previously
 incorporated by reference). The art is familiar with the different
 formulations which can be used as cleaning compositions. In addition to
 typical cleaning compositions, it is readily understood that the protease
 variants of the present invention may be used for any purpose that native
 or wild-type proteases are used. Thus, these variants can be used, for
 example, in bar or liquid soap applications, dishcare formulations,
 contact lens cleaning solutions or products, peptide hydrolysis, waste
 treatment, textile applications, as fusion-cleavage enzymes in protein
 production, etc. The variants of the present invention may comprise
 enhanced performance in a detergent composition (as compared to the
 precursor). As used herein, enhanced performance in a detergent is defined
 as increasing cleaning of certain enzyme sensitive stains such as grass or
 blood, as determined by usual evaluation after a standard wash cycle.
 Proteases of the invention can be formulated into known powdered and liquid
 detergents having pH between 6.5 and 12.0 at levels of about 0.01 to about
 5% (preferably 0.1% to 0.5%) by weight. These detergent cleaning
 compositions can also include other enzymes such as known proteases,
 amylases, cellulases, lipases or endoglycosidases, as well as builders and
 stabilizers.
 The addition of proteases of the invention to conventional cleaning
 compositions does not create any special use limitation. In other words,
 any temperature and pH suitable for the detergent is also suitable for the
 present compositions as long as the pH is within the above range, and the
 temperature is below the described protease's denaturing temperature. In
 addition, proteases of the invention can be used in a cleaning composition
 without detergents, again either alone or in combination with builders and
 stabilizers.
 The present invention also relates to cleaning compositions containing the
 protease variants of the invention The cleaning compositions may
 additionally contain additives which are commonly used in cleaning
 compositions. These can be selected from, but not limited to, bleaches,
 surfactants, builders, enzymes and bleach catalysts. It would be readily
 apparent to one of ordinary skill in the art what additives are suitable
 for inclusion into the compositions. The list provided herein is by no
 means exhaustive and should be only taken as examples of suitable
 additives. It will also be readily apparent to one of ordinary skill in
 the art to only use those additives which are compatible with the enzymes
 and other components in the composition, for example, surfactant.
 When present, the amount of additive present in the cleaning composition is
 from about 0.01% to about 99.9%, preferably about 1% to about 95%, more
 preferably about 1% to about 80%
 The variant proteases of the present invention can be included in animal
 feed such as part of animal feed additives as described in, for example,
 U.S. Pat. Nos. 5,612,055; 5,314,692; and 5,147,642.
 One aspect of the invention is a composition for the treatment of a textile
 that includes variant proteases of the present invention. The composition
 can be used to treat for example silk or wool as described in publications
 such as RD 216,034; EP 134,267; U.S. Pat. No. 4,533,359; and EP 344,259.
 The following is presented by way of example and is not to be construed as
 a limitation to the scope of the claims.
 All publications and patents referenced herein are hereby incorporated by
 reference in their entirety.
 EXAMPLE 1
 A large number of protease variants were produced and purified using
 methods well known in the art. All mutations were made in Bacillus lentus
 GG36 subtilisin. The variants are shown in Table 3.