Non-human Carbonyl hydrolase mutants, DNA sequences and vectors encoding same and hosts transformed with said vectors

Abstract: Novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and DNA sequences encod­ ing the same. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in the amino acid sequ­ ence of a precursor carbonyl hydrolase. Such mutants have one or more properties which are different than the same property of the precursor hydrolase.

Description: The recent development of various in vitro techniques to manipulate the DNA sequences encoding naturally-occuring polypeptides as well as recent developments in the chemical synthesis of relatively short sequences of single and double stranded DNA has resulted in the speculation that such techniques can be used to modify enzymes to improve some functional property in a predictable way. Ulmer, K.M. (1983) Science 219 , 666-671. The only working example disclosed therein is the substitution of a single amino acid within the active site of tyrosyl-tRNA synthetase (Cys35→Ser) which lead to a reduction in enzymatic activity. See Winter, G., et al. (1982) Nature 299 , 756-758; and Wilkinson, A.J., et al. (1983) Biochemistry 22 , 3581-3586 (Cys35→Gly mutation also resulted in decreased activity). When the same t-RNA synthetase was modified by substituting a different amino acid residue within the active site with two different amino acids, one of the mutants (Thr51→Ala) reportedly demonstrated a predicted moderate increase in kcat/Km whereas a second mutant (Thr51→Pro) demonstrated a massive increase in kcat/Km which could not be explained with certainty. Wilkinson, A.H., et al. (1984) Nature 307 , 187-188. Another reported example of a single substitution of an amino acid residue is the substitution of cysteine for isoleucine at the third residue of T4 lysozyme. Perry, L.J., et al. (1984) Science 226 , 555-557. The resultant mutant lysozyme was mildly oxidized to form a disulfide bond between the new cysteine residue at position 3 and the native cysteine at position 97. This crosslinked mutant was initially described by the author as being enzymatically identical to, but more thermally stable than, the wild type enzyme. However, in a "Note Added in Proof", the author indicated that the enhanced stability observed was probably due to a chemical modification of cysteine at residue 54 since the mutant lysozyme with a free thiol at Cys54 has a thermal stability identical to the wild type lysozyme. Similarly, a modified dihydrofolate reductase from E.coli has been reported to be modified by similar methods to introduce a cysteine which could be cross linked with a naturally-occurring cysteine in the reductase. Villafranca, D.E., et al. (1983) Science 222 , 782-788. The author indicates that this mutant is fully reactive in the reduced state but has significantly diminished activity in the oxidized state. In addition, two other substitutions of specific amino acid residues are reported which resulted in mutants which had diminished or no activity. EPO Publication No. 0130756 discloses the substitution of specific residues within B . amyloliquefaciens subtilisin with specific amino acids. Thus, Met222 has been substituted with all 19 other amino acids, Gly166 with 9 different amino acids and Gly169 with Ala and Ser. As set forth below, several laboratories have also reported the use of site directed mutagensis to produce the mutation of more than one amino acid residue within a polypeptide. The amino-terminal region of the signal peptide of the prolipoprotein of the E. coli outer membrane was stated to be altered by the substitution or deletion of residues 2 and 3 to produce a charge change in that region of the polypeptide. Inoyye, S., et al. (1982) Proc. Nat. Acad. Sci. USA 79 , 3438-3441. The same laboratory also reported the substitution and deletion of amino acid redisues 9 and 14 to determine the effects of such substitution on the hydrophobic region of the same signal sequence. Inouye, S., et al. (1984) J. Biol. Chem. 259 , 3729-3733. Double mutants in the active site of tyrosyl-t-RNA synthetase have also been reported. Carter, P.J., et al. (1984) Cell 38 , 835-840. In this report, the improved affinity of the previously described Thr51→Pro mutant for ATP was probed by producing a second mutation in the active site of the enzyme. One of the double mutants, Gly35/Pro51, reportedly demonstrated an unexpected result in that it bound ATP in the transition state better than was expected from the two single mutants. Moreover, the author warns, at least for one double mutant, that it is not readily predictable how one substitution alters the effect caused by the other substitution and that care must be taken in interpreting such substitutions. A mutant is disclosed in U.S. Patent No. 4,532,207, wherein a polyarginine tail was attached to the C-terminal residue of β-urogastrone by modifying the DNA sequence encoding the polypeptide. As disclosed, the polyarginine tail changed the electrophoretic mobility of the urogastrone-polyaginine hybrid permiting selective purification. The polyarginine was subsequently removed, according to the patentee, by a polyarginine specific exopeptidase to produce the purified urogastrone. Properly construed, this reference discloses hybrid polypeptides which do not constitute mutant polypeptides containing the substitution, insertion or deletion of one or more amino acids of a naturally occurring polypeptide. Single and double mutants of rat pancreatic trypsin have also been reported. Craik, C.S., et al . (1985) Science 228 , 291-297. As reported, glycine residues at positions 216 and 226 were replaced with alanine residues to produce three trypsin mutants (two single mutants and one double mutant). In the case of the single mutants, the authors stated expectation was to observe a differential effect on Km. They instead reported a change in specificity (kcat/Km) which was primarily the result of a decrease in kcat. In contrast, the double mutant reportedly demonstrated a differential increase in Km for lysyl and arginyl substrates as compared to wild type trypsin but had virtually no catalytic activity. The references discussed above are provided solely for their disclosure prior to the filing date of the instant case, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications. Based on the above references, however, it is apparent that the modification of the amino acid sequence of wild type enzymes often results in the decrease or destruction of biological activity. Accordingly, it is an object herein to provide carbonyl hydrolase mutants which have at least one property which is different from the same property of the carbonyl hydrolase precursor from which the amino acid of said mutant is derived. It is a further object to provide mutant DNA sequences encoding such carbonyl hydrolase mutants as well as expression vectors containing such mutant DNA sequences. Still further, another object of the present invention is to provide host cells transformed with such vectors as well as host cells which are capable of expressing such mutants either intracellularly or extracellularly. Summary of the Invention The invention includes carbonyl hydrolase mutants, preferably having at least one property which is substantially different from the same property of the precursor non-human carbonyl hydrolase from which the amino acid sequence of the mutant is derived. These properties include oxidative stability, substrate, specificity catalytic activity, thermal stability, alkaline stability, pH activity profile and resistance to proteolytic degradation. The precursor carbonyl hydrolase may be naturally occurring carbonyl hydrolases or recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is derived by the substitution, deletion or insertion of one or more amino acids of the precursor carbonyl hydrolase amino acid sequence. The invention also includes mutant DNA sequences encoding such carbonyl hydrolase mutants. Further the invention includes expression vectors containing such mutant DNA sequences as well as host cells transformed with such vectors which are capable of expressing said carbonyl hydrolase mutants. Brief Description of the Drawings Figure 1 shows the nucleotide sequence of the coding strand, correlated with the amino acid sequence of B . amyloliquefaciens subtilisin gene. Promoter (p) ribosome binding site (rbs) and termination (term) regions of the DNA sequence as well as sequences encoding the presequence (PRE) putative prosequence (PRO) and mature form (MAT) of the hydrolase are also shown. Figure 2 is a schematic diagram showing the substrate binding cleft of subtilisin together with substrate. Figure 3 is a stereo view of the S-1 binding subsite of B . amyloliquefaciens subtilisin showing a lysine P-1 substrate bound in the site in two different ways. Figure 3A shows Lysine P-1 substrate bound to form a salt bridge with a Glu at position 156. Figure 3B shows Lysine P-1 substrate bound to form a salt bridge with Glu at position 166. Figure 4 is a schematic diagram of the active site of subtilisin Asp32, His64 and Ser221. Figures 5A and 5B depict the amino acid sequence of subtilisin obtained from various sources. The residues directly beneath each residue of B . amyloliquefaciens subtilisin are equivalent residues which (1) can be mutated in a similar manner to that described for B . amyloliquefaciens subtilisin, or (2) can be used as a replacement amino acid residue in B . amyloliquefaciens subtilisin. Figure 5C depicts conserved residues of B . amyloliquefaciens subtilisin when compared to other subtilisin sequences. Figures 6A and 6B depict the inactivation of the mutants Met222L and Met222Q when exposed to various organic oxidants. Figure 7 depicts the ultraviolet spectrum of Met222F subtilisin and the difference spectrum generated after inactivation by diperdodecanoic acid (DPDA). Figure 8 shows the pattern of cyanogen bromide digests of untreated and DPDA oxidized subtilisin Met222F on high resolution SDS-pyridine peptide gels. Figure 9 depicts a map of the cyanogen bromide fragments of Fig. 8 and their alignment with the sequence of subtilisin Met222F. Figure 10 depicts the construction of mutations between codons 45 and 50 of B . amyloliquefaciens subtilisin. Figure 11 depicts the construction of mutations between codons 122 and 127 of B . amyloliquefaciens subtilisin. Figure 12 depicts the effect of DPDA on the activity of subtilisin mutants at positions 50 and 124 in subtilisin Met222F. Figure 13 depicts the construction of mutations at codon 166 of B . amyloliquefaciens subtilisin. Figure 14 depicts the effect of hydrophobicity of the P-1 substrate side-chain on the kinetic parameters of wild-type B . amyloliquefaciens subtilisin. Figure 15 depicts the effect of position 166 side-chain substitutions on P-l substrate specificity. Figure 15A shows position 166 mutant subtilisins containing non-branched alkyl and aromatic side-chain substitutions arranged in order of increasing molecular volume. Figure 15B shows a series of mutant enzymes progressing through β- and γ-branched aliphatic side chain substitutions of increasing molecular volume. Figure 16 depicts the effect of position 166 side-chain volumn on log kcat/Km for various P-1 substrates. Figure 17 shows the substrate specificity differences between Ile166 and wild-type (Gly166) B . amyloliquefaciens subtilisin against a series of alphatic and aromatic substrates. Each bar represents the difference in log kcat/Km for Ile166 minus wild-type (Gly166) subtilisin. Figure 18 depicts the construction of mutations at codon 169 of B . amyloliquefaciens subtilisin. Figure 19 depicts the construction of mutations at codon 104 of B . amyloliquefaciens subtilisin. Figure 20 depicts the construction of mutations at codon 152 B . amyloliquefaciens subtilisin. Figure 21 depicts the construction of single mutations at codon 156 and double mutations at codons 156 and 166 of B . amyloliquefaciens subtilisin. Figure 22 depicts the construction of mutations at codon 217 for B . amyloliquefaciens subtilisin. Figure 23 depicts the kcat/Km versus pH profile for mutations at codon 156 and 166 in B . amyloliquefaciens subtilisin. Figure 23A depicts the kcat/Km versus pH profile for mutations at codon 156 and 166 in B . amyloliquefaciens subtilisin. Figure 24 depicts the kcat/Km versus pH profile for mutations at codon 222 in B . amyloliquefaciens subtilisin. Figure 25 depicts the constructing mutants at codons 94, 95 and 96. Figures 26 and 27 depict substrate specificity of various wild type and mutant subtilisins for different substrates. Figures 28 A, B, C and D depict the effect of charge in the P-1 binding sites due to substitutions at codon 156 and 166. Figures 29 A and B are a stereoview of the P-1 binding site of subtilisin BPN' showing a lysine P-1 substrate bound in the site in two ways. In 29A, Lysine P-1 substrate is built to form a salt bridge with a Glu at codon 156. In 29B, Lysine P-1 substrate is built to form a salt bridge with Glu at codon 166. Figure 30 demonstrates residual enzyme activity versus temperature curves for purified wild-type (Panel A), C22/C87 (Panel B) and C24/C87 (Panel C). Figure 31 depicts the strategy for producing point mutations in the subtilisin coding sequence by misincorporation of α -thioldeoxynucleotide triphosphates. Figure 32 depicts the autolytic stability of purified wild type and mutant subtilisins 170E, 107V, 213R and 107V/213R at alkaline pH. Figure 33 depicts the autolytic stability of purified wild type and mutant subtilisins V50, F50 and F50/V107/R213 at alkaline pH. Figure 34 depicts the strategy for constructing plasmids containing random cassette mutagenesis over residues 197 through 228. Figure 35 depicts the oligodeoxynucleotides used for random cassette mutagenesis over residues 197 through 228. Figure 36 depicts the construction of mutants at codon 204. Figure 37 depicts the oligodeoxynucleotides used for synthesizing mutants at codon 204. Detailed Description The inventors have discovered that various single and multiple in vitro mutations involving the substitution, deletion or insertion of one or more amino acids within a non-human carbonyl hydrolase amino acid sequence can confer advantageous properties to such mutants when compared to the non-mutated carbonyl hydrolase. Specifically, B . amyloliquefaciens subtilisin, an alkaline bacterial protease, has been mutated by modifying the DNA encoding the subtilisin to encode the substitution of one or more amino acids at various amino acid residues within the mature form of the subtilisin molecule. These in vitro mutant subtilisins have at least one property which is different when compared to the same property of the precursor subtilisin. These modified properties fall into several categories including: oxidative stability, substrate specificity, thermal stability, alkaline stability, catalytic activity, pH activity profile, resistance to proteolytic degradation, Km, kcat and Km/kcat ratio. Carbonyl hydrolases are enzymes which hydrolyze compounds containing bonds in which X is oxygen or nitrogen. They include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. Naturally occurring carbonyl hydrolases principally include hydrolases, e.g. lipases and peptide hydrolases, e.g. subtilisins or metalloproteases. Peptide hydrolases include α-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 exoproteases. "Recombinant carbonyl hydrolase" refers to a carbonyl hydrolase in which the DNA sequence encoding the naturally occurring carbonyl hydrolase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the carbonyl hydrolase amino acid sequence. Suitable modification methods are disclosed herein and in EPO Publication No. 0130756 published January 9, 1985. Subtilisins are bacterial carbonyl hydrolases 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 bacterial 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-histidineserine. 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. "Recombinant subtilisin" refers to a subtilisin in which the DNA sequence encoding the subtilisin is modified to produce a mutant DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally occurring subtilisin amino acid sequence. Suitable methods to produce such modification include those disclosed herein and in EPO Publication No. 0130756. For example, the subtilisin multiple mutant herein containing the substitution of methionine at amino acid residues 50, 124 and 222 with phenylalanine, isoleucine and glutamine, respectively, can be considered to be derived from the recombinant subtilisin containing the substitution of glutamine at residue 222 (Q222) disclosed in EPO Publication No. 0130756. The multiple mutant thus is produced by the substitution of phenylalanine for methionine at residue 50 and isoleucine for methionine at residue 124 in the Q222 recombinant subtilisin. "Carbonyl hydrolases" and their genes 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 carbonyl hydrolase and their genes may be obtained include yeast such as S . cerevisiae , fungi such as Aspergillus sp., and non-human mammalian sources such as, for example, Bovine sp. from which the gene encoding the carbonyl hydrolase chymosin can be obtained. As with subtilisins, a series of carbonyl hydrolases can be obtained from various related species which have amino acid sequences which are not entirely homologous between the members of that series but which nevertheless exhibit the same or similar type of biological activity. Thus, non-human carbonyl hydrolase as used herein has a functional definition which refers to carbonyl hydrolases which are associated, directly or indirectly, with procaryotic and non-human eucaryotic sources. A "carbonyl hydrolase mutant" has an amino acid sequence which is derived from the amino acid sequence of a non-human "precursor carbonyl hydrolase". The precursor carbonyl hydrolases include naturally-occurring carbonyl hydrolases and recombinant carbonyl hydrolases. The amino acid sequence of the carbonyl hydrolase mutant is "derived" from the precursor hydrolase 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 carbonyl hydrolase rathern than manipulation of the precursor carbonyl hydrolase per se . Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in EPO Publication No. 0130756. Specific residues of B . amyloliquefaciens subtilisin are identified for substitution, insertion or deletion. These amino acid position numbers refer to those assigned to the B . amyloliquefaciens subtilisin sequence presented in Fig. 1. The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor carbonyl hydrolases containing amino acid residues which are "equivalent" to the particular identified residues in B . amyloliquefaciens subtilisin. A residue (amino acid) of a precursor carbonyl hydrolase is equivalent to a residue of B . amyloliquefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analagous to a specific residue or portion of that residue in B . 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 carbonyl hydrolase is directly comparted to the B . amyloliquefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in all subtilisins for which sequence is known (Figure 5C). 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 B . 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. For example, in Figure 5A the amino acid sequence of subtilisin from B . amyloliquefaciens B . subtilisin var. I168 and B . lichenformis (carlsbergensis) 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 residues are identified in Fig. 5C. These conserved residues thus may be used to define the corresponding equivalent amino acid residues of B . amyloliquefaciens subtilisin in other carbonyl hydrolases such as thermitase derived from Thermoactinomyces. These two particular sequences are aligned in Fig. 5B to produce the maximum homology of conserved residues. As can be seen there are a number of insertions and deletions in the thermitase sequence as compared to B . amyloliquefaciens subtilisin. Thus, in thermitase the equivalent amino acid of Tyr217 in B . amyloliquefaciens subtilisin is the particular lysine shown beneath Tyr217. In Fig. 5A, the equivalent amino acid at position 217 in B . amyloliquefaciens subtilisin is Tyr. Likewise, in B . subtilis subtilisin position 217 is also occupied by Tyr but in B . licheniformis position 217 is occupied by Leu. Thus, these particular residues in thermitase, and subtilisin from B . subtilisin and B . licheniformis may be substituted by a different amino acid to produce a mutant carbonyl hydrolase since they are equivalent in primary structure to Tyr217 in B . amyloliquefaciens subtilisin. Equivalent amino acids of course are not limited to those for Tyr217 but extend to any residue which is equivalent to a residue in B . amyloliquefaciens whether such residues are conserved or not. Equivalent residues homologous at the level of tertiary structure for a precursor carbonyl hydrolase whose tertiary structure has been determined by x-ray crystallography, are defined as those for which the atomic coordinates of 2 or more of the main chain atoms of a particular amino acid residue of the precursor carbonyl hydrolase and B . amyloliquefaciens subtilisin (N on N, CA on CA, C on C, and O on O) are within 0.13nm and preferably 0.1nm 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 carbonyl hydrolase in question to the B . amyloliquefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. Equivalent residues which are functionally analogous to a specific residue of B . amyloliquefaciens subtilisin are defined as those amino acids of the precursor carbonyl hydrolases 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 B . amyloliquefaciens subtilisin as described herein. Further, they are those residues of the precursor carbonyl hydrolase (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 0.13nm of the corresponding side chain atoms of B . amyloliquefaciens subtilisin. The three dimensional structures would be aligned as outlined above. Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a mutant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in a naturally occurring sequence. The carbonyl hydrolase mutants of the present invention include the mature forms of carbonyl hydrolase mutants as well as the pro- and prepro-forms of such hydrolase mutants. The prepro-forms are the preferred construction since this facilitates the expression, secretion and maturation of the carbonyl hydrolase mutants. "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 EPO Publication No. 0130756 to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing subtilisin 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 EPO Publicatin No. 0130756 and further described by Yang, M.Y., et al. (1984) J. Bacteriol. 160 , 15-21. Other host cells for expressing subtilisin include Bacillus subtilis I168 (EPO Publication No. 0130756). 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 carbonyl hydrolase mutants or expressing the desired carbonyl hydrolase mutant. In the case of vectors which encode the pre or prepro form of the carbonyl hydrolase mutant, such mutants, 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 carbonyl hydrolase may be obtained in accord with the general methods described herein in EPO publication No. 0130756. Once the carbonyl hydrolase gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor carbonyl hydrolase. Such modifications include the production of recombinant carbonyl hydrolases as disclosed in EPO Publication No. 0130756 and the production of carbonyl hydrolase mutants described herein. The carbonyl hydrolase mutants of the present invention may be generated by site specific mutagenesis (Smith, M. (1985) Ann, Rev. Genet. 423 ; Zoeller, M.J., et al. (1982) Nucleic Acid Res. 10 , 6487-6500), cassette mutagenesis (EPO Publication No. 0130756) or random mutagenesis (Shortle, D., et al. (1985) Genetics , 110 , 539; Shortle, D., et al. (1986) Proteins: Structure, Function and Genetics , 1 , 81; Shortle, D. (1986) J. Cell. Biochem , 30 , 281; Alber, T., et al. (1985) Proc. Natl. Acad. of Sci. , 82 , 747; Matsumura, M., et al. (1985) J. Biochem. , 260 , 15298; Liao, H., et al. (1986) Proc. Natl. Acad. of Sci. , 83 576) of the cloned precursor carbonyl hydrolase. Cassette mutagenesis and the random mutagenesis method disclosed herein are preferred. The mutant carbonyl hydrolases expressed upon transformation of suitable hosts are screened for enzymes exhibiting one or more properties which are substantially different from the properties of the precursor carbonyl hydrolases, e.g., changes in substrate specificity, oxidative stability, thermal stability, alkaline stability, resistance to proteolytic degradation, pH-activity profiles and the like. A change in substrate specificity is defined as a difference between the kcat/Km ratio of the precursor carbonyl hydrolase and that of the hydrolase mutant. The kcat/Km ratio is a measure of catalytic efficienty. Carbonyl hydrolase mutants with increased or diminished kcat/Km ratios are described in the examples. Generally, the objective will be to secure a mutant having a greater (numerically large) kcat/Km ratio for a given substrate, thereby enabling the use of the enzyme to more efficiently act on a target substrate. A substantial change in kcat/Km ratio is preferably at least 2-fold increase or decrease. However, smaller increases or decreases in the ratio (e.g., at least 1.5-fold) are also considered substantial. An increase in kcat/Km ratio for one substrate may be accompanied by a reduction in kcat/Km ratio for another substrate. This is a shift in substrate specificity, and mutants exhibiting such shifts have utility where the precursor hydrolase is undesirable, e.g. to prevent undesired hydrolysis of a particular substrate in an admixture of substrates. Km and kcat are measured in accord with known procedures, as described in EPO Publication No. 0130756 or as described herein. Oxidative stability is measured either by known procedures or by the methods described hereinafter. A substantial change in oxidative stability is evidenced by at least about 50% increase or decrease (preferably decrease) in the rate of loss of enzyme activity when exposed to various oxidizing conditions. Such oxidizing conditions are exposure to the organic oxidant diperdodecanoic acid (DPDA) under the conditions described in the examples. Alkaline stability is measured either by known procedures or by the methods described herein. A substantial change in alkaline stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half life of the enzymatic activity of a mutant when compared to the precursor carbonyl hydrolase. In the case of subtilisins, alkaline stability was measured as a function of autoproteolytic degradation of subtilisin at alkaline pH, e.g. for example, 0.1M sodium phosphate, pH 12 at 25° or 30°C. Thermal stability is measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced by at least about a 5% or greater increase or decrease (preferably increase) in the half-life of the catalytic activity of a mutant when exposed to a relatively high temperature and neutral pH as compared to the precursor carbonyl hydrolase. In the case of subtilisins, thermal stability is measured by the autoproteolytic degradation of subtilisin at elevated temperatures and neutral pH, e.g., for example 2mM calcium chloride, 50mM MOPS pH 7.0 at 59°C. The inventors have produced mutant subtilisins containing the substitution of the amino acid residues of B . amyloliquefaciens subtilisin shown in Table I. The wild type amino acid sequence and DNA sequence of B . amyloliquefaciens subtilisin is shown in Fig. 1. Residue Replacement Amino Acid Tyr21 F A Thr22 C Ser24 C Asp32 Q S Ser33 A T Asp36 A G Gly46 V Ala48 E V R Ser49 C L Met50 C F V Asn77 D Ser87 C Lys94 C Val95 C Leu96 D Tyr104 A C D E F G H I K L M N P Q R S T V W Ile107 V Gly110 C R Met124 I L Asn155 A D H Q T Glu156 Q S Gly166 C E I L M P S T W Y Gly169 C D E F H I K L M N P Q R T V W Y Lys170 E R Tyr171 F Pro172 E Q Phe189 A C D E G H I K L M N P Q R S T V W Y Asp197 R A Met199 I Ser204 C R L P Lys213 R T Tyr217 A C D E F G H I K L M N P Q R S T V W Ser221 A C The different amino acids substituted are represented in Table I by the following single letter designations: Amino acid or residue thereof 3-letter symbol 1-letter symbol Alanine Ala A Glutamate Glu E Glutamine Gln Q Aspartate Asp D Asparagine Asn N Leucine Leu L Glycine Gly G Lysine Lys K Serine Ser S Valine Val V Arginine Arg R Threonine Thr T Proline Pro P Isoleucine Ile I Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y Cysteine Cys C Tryptophan Trp W Histidine His H Except where otherwise indicated by context, wild-type amino acids are represented by the above three-letter symbols and replaced amino acids by the above single-letter symbols. Thus, if the methionine at residue 50 in B . amyloliquefaciens subtilisin is replaced by phenylalanine, this mutation (mutant) may be designated Met50F or F50. Similar designations are used for multiple mutants. In addition to the amino acids used to replace the residues disclosed in Table I, other replacements of amino acids at these residues are expected to produce mutant subtilisins having useful properties. These residues and replacement amino acids are shown in Table II. Residue Replacement Amino Acid(s) Tyr-21 L Thr22 K Ser24 A Asp32 Ser33 G Gly46 Ala48 Ser49 Met50 L K I V Asn77 D Ser87 N Lys94 R Q Val95 L I Tyr104 Met124 K A Ala152 C L I T M Asn155 Glu156 A T M L Y Gly166 Gly169 Tyr171 K R E Q Pro172 D N Phe189 Tyr217 Ser221 Met222 Each of the mutant subtilisins in Table I contain the replacement of a single residue of the B . amyloliquefaciens amino acid sequence. These particular residues were chosen to probe the influence of such substitutions on various properties of B . amyloliquefacien subtilisin. Thus, the inventors have identified Met124 and Met222 as important residues which if substituted with another amino acid produce a mutant subtilisin with enhanced oxidative stability. For Met124, Leu and Ile are preferred replacement amino acids. Preferred amino acids for replacement of Met222 are disclosed in EPO Publication No. 0130756. Various other specific residues have also been identified as being important with regard to substrate specificity. These residues include Tyr104, Ala152, Glu156, Gly166, Gly169, Phe189 and Tyr217 for which mutants containing the various replacement amino acids presented in Table I have already been made, as well as other residues presented below for which mutants have yet to be made. The identification of these residues, including those yet to be mutated, is based on the inventors' high resolution crystal structure of B . amyloliquefaciens subtilisin to 1.8 A (see Table III), their experience with in vitro mutagenesis of subtilisin and the literature on subtilisin. This work and the x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J.D., et al . (1972) Biochemistry 11 , 2439-2449), product complexes (Robertus, J.D., et al . (1972) Biochemistry 11 , 4293-4303), and transition state analogs (Matthews, D.A., et al (1975) J. Biol. Chem. 250 , 7120-7126; Poulos, T.L., et al . (1976) J. Biol. Chem. 251 , 1097-1103), has helped in identifying an extended peptide binding cleft in subtilisin. This substrate binding cleft together with substrate is schematically diagramemed in Fig. 2, according to the nomenclature of Schechter, I., et al . (1967) Biochem Bio. Res. Commun. 27 , 157. The scissile bond in the substrate is identified by an arrow. The P and P' designations refer to the amino acids which are positioned respectively toward the amino or carboxy terminus relative to the scissle bond. The S and S' designations refer to subsites in the substrate binding cleft of subtilisin which interact with the corresponding substrate amino acid residues. The above structural studies together with the kinetic data presented herein and elsewhere (Philipp, M., et al. (1983) Mol. Cell. Biochem. 51 , 5-32; Svendsen, I.B. (1976) Carlsberg Res. Comm. 41 , 237-291; Markland, S.F. Id ; Stauffe, D.C., et al. (1965) J. Biol. Chem. 244 , 5333-5338) indicate that the subsites in the binding cleft of subtilisin are capable of interacting with substrate amino acid residues from P-4 to P-2'. The most extensively studied of the above residues are Gly166, Gly169 and Ala152. These amino acids were identified as residues within the S-1 subsite. As seen in Fig. 3, which is a stereoview of the S-1 subsite, Gly166 and Gly169 occupy positions at the bottom of the S-1 subsite, whereas Ala152 occupies a position near the top of S-1, close to the catalytic Ser221. All 19 amino acid substitutions of Gly166 and Gly169 have been made. As will be indicated in the examples which follow, the preferred replacement amino acids for Gly166 and/or Gly169 will depend on the specific amino acid occupying the P-1 position of a given substrate. The only substitutions of Ala152 presently made and analyzed comprise the replacement of Ala152 with Gly and Ser. The results of these substitutions on P-1 specificity will be presented in the examples. In addition to those residues specifically associated with specificity for the P-1 substrate amino acid, Tyr104 has been identified as being involved with P-4 specificity. Substitutions at Phe189 and Tyr217, however, are expected to respectively effect P-2' and P-1' specificity. The catalytic activity of subtilisin has also been modified by single amino acid substitutions at Asn155. The catalytic triad of subtilisin is shown in Fig. 4. As can be seen, Ser221, His64 and Asp32 arc positioned to facilitate nucleophilic attach by the serine hydoxylate on the carbonyl of the scissile peptide bond. Crystallographic studies of subtilisin (Robertus, et al . (1972) Biochem. 11 , 4293-4303; Matthews, et al . (1975) J. Biol. Chem. 250 , 7120-7126; Poulos, et al . (1976) J. Biol. Chem. 250 , 1097-1103) show that two hydrogen bonds are formed with the oxyanion of the substrate transition state. One hydrogen bond donor is from the catalytic serine-221 main-chain amide while the other is from one of the NE2 protons of the asparagine-155 side chain. See Fig. 4. Asn155 was substituted with Ala, Asp, His, Glu and Thr. These substitutions were made to investigate the the stabilization of the charged tetrahedral intermediate of the transition state complex by the potential hydrogen bond between the side chain of Asn155 and the oxyanion of the intermediate. These particular substitutions caused large decreases in substrate turnover, kcat (200 to 4,000 fold), marginal decreases in substrate binding Km (up to 7 fold), and a loss in transition state stabilization energy of 2.2 to 4.7 kcal/mol. The retention of Km and the drop in kcat will make these mutant enzymes useful as binding proteins for specific; peptide sequences, the nature of which will be determined by the specificity of the precursor protease. Various other amino acid residues have been identified which affect alkaline stability. In some cases, mutants having altered alkaline stability also have altered thermal stability. In B amyloliquefaciens subtilisin residues Asp36, Ile107, Lys170, Ser204 and Lys213 have been identified as residues which upon substitution with a different amino acid alter the alkaline stability of the mutated enzyme as compared to the precursor enzyme. The substitution of Asp36 with Ala and the substitution of Lys170 with Glu each resulted in a mutant enzyme having a lower alkaline stability as compared to the wild type subtilisin. When Ile107 was substituted with Val, Ser204 substituted with Cys, Arg or Leu or Lys213 substituted with Arg, the mutant subtilisin had a greater alkaline stability as compared to the wild type subtilisin. However, the mutant Ser204P demonstrated a decrease in alkaline stability. In addition, other residues, identified as being associated with the modification of other properties of subtilisin, also affect alkaline stability. These residues include Ser24, Met50, Glu156, Gly166, Gly169 and Tyr217. Specifically the following particular substitutions result in an increased alkaline stability: Ser24C, Met50F, Gly156Q or S, Gly166A, H, K, N or Q, Gly169S or A, and Tyr217F, K, R or L. The mutant Met50V, on the other hand, results in a decrease in the alkaline stability of the mutant subtilisin as compared to wild type subtilisin. Other residues involved in alkaline stability based on the alkaline stability screen include Asp197 and Met222. Particular mutants include Asp197(R or A) and Met 222 (all other amino acids). Various other residues have been identified as being involved in thermal stability as determined by the thermal stability screen herein. These residues include the above identified residues which effect alkaline stability and Met199 and Tyr21. These latter two residues are also believed to be important for alkaline stability. Mutants at these residues include I199 and F21. The amino acid sequence of B . amyloliquefaciens substilisin has also been modified by substituting two or more amino acids of the wild-type sequence. Six categories of multiply substituted mutant subtilisin have been identified. The first two categories comprise thermally and oxidatively stable mutants. The next three other categories comprise mutants which combine the useful properties of any of several single mutations of B . amyloliquefaciens subtilisin. The last category comprises mutants which have modified alkaline and/or thermal stability. The first category comprises double mutants in which two cysteine residues have been substituted at various amino acid residue positions within the subtilisin molecule. Formation of disulfide bridges between the two substituted cysteine residues results in mutant subtilisins with altered thermal stability and catalytic activity. These mutants include A21/C22/C87 and C24/C87 which will be described in more detail in Example 11. The second category of multiple subtilisin mutants comprises mutants which are stable in the presence of various oxidizing agents such as hydrogen peroxide or peracids. Examples 1 and 2 describe these mutants which include F50/I124/Q222, F50/I124, F50/Q222, F50/L124/Q222, I124/Q222 and L124/Q222. The third category of multiple subtilisin mutants comprises mutants with substitutions at position 222 combined with various substitutions at positions 166 or 169. These mutants, for example, combine the property of oxidative stability of the A222 mutation with the altered substrate specificity of the various 166 or 169 substitutions. Such multiple mutants include A166/A222, A166/C222, F166/C222, K166/A222, K166/C222, V166/A222 and V166/C222. The K166/A222 mutant subtilisin, for example, has a kcat/Km ratio which is approximately two times greater than that of the single A222 mutant subtilisin when compared using a substrate with phenylalanine as the P-1 amino acid. This category of multiple mutant is described in more detail in Example 12. The fourth category of multiple mutants combines substitutions at position 156 (Glu to Q or S) with the substitution of Lys at position 166. Either of these single mutations improve enzyme performance upon substrates with glutamate as the P-1 amino acid. When these single mutations are combined, the resulting multiple enzyme mutants perform better than either precursor. See Example 9. The fifth category of multiple mutants contain the substitution of up to four amino acids of the B . amyloliquefaciens subtilisin sequence. These mutants have specific properties which are virtually identicle to the properties of the subtilisin from B . licheniformis . The subtilisin from B . licheniformis differs from B . amyloliquefaciens subtilisin at 87 out of 275 amino acids. The multiple mutant F50/S156/A169/L217 was found to have similar substrate specificity and kinetics to the licheniformis enzyme. (See Example 13.) However, this is probably due to only three of the mutations (S156, A169 and L217) which are present in the substrate binding region of the enzyme. It is quite surprising that, by making only three changes out of the 87 different amino acids between the sequence of the two enzymes, the B . amyloliquifaciens enzyme was converted into an enzyme with properties similar to B . licheniformis enzyme. Other enzymes in this series include F50/Q156/N166/L217 and F50/S156/L217. The sixth category of multiple mutants includes the combination of substitutions at position 107 (Ile to V) with the substitution of Lys at position 213 with Arg, and the combination of substitutions of position 204 (preferably Ser to C or L but also to all other amino acids) with the substituion of Lys at position 213 with R. Other multiple mutants which have altered alkaline stability include Q156/K166, Q156/N166, S156/K166, S156/N166 (previously identified as having altered substrate specificity), and F50/S156/A169/L217 (previously identified as a mutant of B . amyloliquifaciens subtilisin having properties similar to subtilisin from B . licheniformis) . The mutant F50/V107/R213 was constructed based on the observed increase in alkaline stability for the single mutants F50, V107 and R213. It was determined that the V107/R213 mutant had an increased alkaline stability as compared to the wild type subtilisin. In this particular mutant, the increased alkaline stability was the result of the cumulative stability of each of the individual mutations. Similarly, the mutant F50/V107/R213 had an even greater alkaline stability as compared to the V107/R213 mutant indicating that the increase in the alkaline stability due to the F50 mutation was also cumulative. Table IV summarizes the multiple mutants which have been made including those not mentioned above. In addition, based in part on the above results, substitution at the following residues in subtilisin is expected to produce a multiple mutant having increased thermal and alkaline stability: Ser24, Met50, Ile107, Glu156, Gly166, Gly169, Ser204, Lys213, Gly215, and Tyr217. Double Mutants Triple, Quadruple or Other Multiple C22/C87 F50/I124/Q222 C24/C87 F50/L124/Q222 V45/V48 F50/L124/A222 C49/C94 A21/C22/C87 C49/C95 F50/S156/N166/L217 C50/C95 F50/Q156/N166/L217 C50/C110 F50/S156/A169/L217 F50/I124 F50/S156/L217 F50/Q222 F50/Q156/K166/L217 I124/Q222 F50/S156/K166/L217 Q156/D166 F50/Q156/K166/K217 Q156/K166 F50/S156/K166/K217 Q156/N166 F50/V107/R213 S156/D166 [S153/S156/A158/G159/S160/Δ161-164/I165/S166/A169/R170] S156/K166 S156/N166 L204/R213 S156/A169 R213/204A, E, Q, D, N, G, K, V, R, T, P, I, M, F, Y, W or H A166/A222 A166/C222 F166/A222 V107/R213 F166/C222 K166/A222 K166/C222 V166/A222 V166/C222 A169/A222 A169/A222 A169/C222 A21/C22 In addition to the above identified amino acid residues, other amino acid residues of subtilisin are also considered to be important with regard to substrate specificity. Mutation of each of these residues is expected to produce changes in the substrate specificity of subtilisin. Moreover, multiple mutations among these residues and among the previously identified residues are also expected to produce subtilisin mutants having novel substrate specificity. Particularly important residues are His67, Ile107, Leu126 and Leu135. Mutation of His67 should alter the S-1' subsite, thereby altering the specificity of the mutant for the P-1' substrate residue. Changes at this position could also affect the pH activity profile of the mutant. This residue was identified based on the inventor's substrate modeling from product inhibitor complexes. Ile107 is involved in P-4 binding. Mutation at this position thus should alter specificity for the P-4 substrate residue in addition to the observed effect on alkaline stability. Ile107 was also identified by molecular modeling from product inhibitor complexes. The S-2 binding site includes the Leu126 residue. Modification at this position should therefore affect P-2 specificity. Moreover, this residue is believed to be important to convert subtilisin to an amino peptidase. The pH activity profile should also be modified by appropriate substitution. These residues were identified from inspection of the refined model, the three dimensional structure from modeling studies. A longer side chain is expected to preclude binding of any side chain at the S-2 subsite. Therefore, binding would be restricted to subsites S-1, S-1', S-2', S-3' and cleavage would be forced to occur after the amino terminal peptide. Leu135 is in the S-4 subsite and if mutated should alter substrate specificity for P-4 if mutated. This residue was identified by inspection of the three-dimensional structure and modeling based on the product inhibitor complex of F222. In addition to theses sites, specific amino acid residues within the segments 97-103, 126-129 and 213-215 are also believed to be important to substrate binding. Segments 97-103 and 126-129 form an antiparallel beta sheet with the main chain of substrate residues P-4 through P-2. Mutating residues in those regions should affect the substrate orientation through main chain (enzyme) - main chain (substrate) interactions, since the main chain of these substrate residues do not interact with these particular residues within the S-4 through S-2 subsites. Within the segment 97-103, Gly97 and Asp99 may be mutated to alter the position of residues 101-103 within the segment. Changes at these sites must be compatible, however. In B . amyloliquifaciens subtilisin Asp99 stabilizes a turn in the main chain tertiary folding that affects the direction of residues 101-103. B . licheniformis subtilisin Asp97, functions in an analogous manner. In addition to Gly97 and Asp99, Ser101 interacts with Asp99 in B . amyliquefaciens subtilisin to stabilize the same main chain turn. Alterations at this residue should alter the 101-103 main chain direction. Mutations at Glu103 are also expected to affect the 101-103 main chain direction. The side chain of Gly102 interacts with the substrate P-3 amino acid. Side chains of substituted amino acids thus are expected to significantly affect specificity for the P-3 substrate amino acids. All the amino acids within the 127-129 segment are considered important to substrate specificity. Gly127 is positioned such that its side chain interacts with the S-1 and S-3 subsites. Altering this residue thus should alter the specificity for P-1 and P-3 residues of the substrate. The side chain of Gly128 comprises a part of both the S-2 and S-4 subsites. Altered specificity for P-2 and P-4 therefore would be expected upon mutation. Moreover, such mutation may convert subtilisin into an amino peptidase for the same reasons substitutions of Leu126 would be expected to produce that result. The Pro129 residue is likely to restrict the conformational freedom of the sequence 126-133, residues which may play a major role in determining P-1 specificity. Replacing Pro may introduce more flexibility thereby broadening the range of binding capabilities of such mutants. The side chain of Lys213 is located within the S-3 subsite. All of the amino acids within the 213-215 segment are also considered to be important to substrate specificity. Accordingly, altered P-3 substrate specificity is expected upon mutation of this residue. The Tyr214 residue does not interact with substrate but is positioned such that it could affect the conformation of the hair pin loop 204-217. Finally, mutation of the Gly215 residue should affect the S-3' subsite, and thereby alter P-3' specificity. In addition to the above substitutions of amino acids, the insertion or deletion of one or more amino acids within the external loop comprising residues 152-172 may also affect specificity. This is because these residues may play a role in the "secondary contact region" described in the model of streptomyces subtilisin inhibitor complexed with subtilisin. Hirono, et al . (1984) J. Mol. Biol. 178 , 389-413. Thermitase K has a deletion in this region, which eliminates several of these "secondary contact" residues. In particular, deletion of residues 161 through 164 is expected to produce a mutant subtilisin having modified substrate specificity. In addition, a rearrangement in this area induced by the deletion should alter the position of many residues involved in substrate binding, predominantly at P-1. This, in turn, should affect overall activity against proteinaceous substrates The effect of deletion of residues 161 through 164 has been shown by comparing the activity of the wild type (WT) enzyme with a mutant enzyme containing this deletion as well as multiple substitutions (i.e., S153/S156/A158/G159/S160/Δ161-164/I165/S166/A169/R170). This produced the following results: kcat Km kcat/Km WT 50 1.4x10⁻⁴ 3.6x10⁵ Deletion mutant 8 5.0x10⁻⁶ 1.6x10⁶ The WT has a kcat 6 times greater than the deletion mutant but substrate binding is 28 fold tighter by the deletion mutant. The overall efficiency of the deletion mutant is thus 4.4 times higher than the WT enzyme. All of these above identified residues which have yet to be substituted, deleted or inserted into are presented in Table VI. Substitution/Insertion/Deletion Residues His67 Ala152 Leu126 Ala153 Leu135 Gly154 Gly97 Asn155 Asp99 Gly156 Ser101 Gly157 Gly102 Gly160 Glu103 Thr158 Leu126 Ser159 Gly127 Ser161 Gly128 Ser162 Pro129 Ser163 Tyr214 Thr164 Gly215 Val165 Gly166 Gly169 Tyr167 Lys170 Pro168 Tyr171 Pro172 The following disclosure is intended to serve as a representation of embodiments herein, and should not be construed as limiting the scope of this application. These specific examples disclose the construction of certain of the above identified mutants. The construction of the other mutants, however, is apparent from the disclosure herein and that presented in EPO Publication No. 0130756. All literature citations are expressly incorporated by reference. EXAMPLE 1 Identification of Peracid Oxidizable Residues of Subtilisin Q222 and L222 As shown in Figures 6A and 6B, organic peracid oxidants inactivate the mutant subtilisins Met222L and Met222Q (L222 and Q222). This example describes the identification of peracid oxidizable sites in these mutant subtilisins. First, the type of amino acid involved in peracid oxidation was determined. Except under drastic conditions (Means, G.E., et al. (1971) Chemical Modifications of Proteins , Holden-Day, S.F., CA, pp. 160-162), organic peracids modify only methionine and tryptophan in subtilisin. Difference spectra of the enzyme over the 250nm to 350nm range were determined during an inactivation titration employing the reagent, diperdodecanoic acid (DPDA) as oxidant. Despite quantitative inactivation of the enzyme, no change in absorbance over this wavelength range was noted as shown in Figures 7A and 7B indicating that tryptophan was not oxidized. Fontana, A., et al. (1980) Methods in Peptide and Protein Sequence Analysis (C. Birr ed.) Elsevier, New York, p. 309. The absence of tryptophan modification implied oxidation of one or more of the remaining methionines of B . amyloliquefaciens subtilisin. See Figure 1. To confirm this result the recombinant subtilisin Met222F was cleaved with cyanogen bromide (CNBr) both before and after oxidation by DPDA. The peptides produced by CNBr cleavage were analyzed on high resolution SDS-pyridine peptide gels (SPG). Subtilisin Met222F (F222) was oxidized in the following manner. Purified F222 was resuspended in 0.1 M sodium borate pH 9.5 at 10 mg/ml and was added to a final concentration of 26 diperdodecanoic acid (DPDA) at 26 mg/ml was added to produce an effective active oxygen concentration of 30 ppm. The sample was incubated for at least 30 minutes at room temperature and then quenched with 0.1 volume of 1 M Tris pH 8.6 buffer to produce a final concentration of 0.1 M Tris pH 8.6). 3mM phenylmethylsulfonyl fluoride (PMSF) was added and 2.5 ml of the sample was applied to a Pharmacia PD10 column equilibrated in 10 mM sodium phosphate pH 6.2, 1 mM PMSF. 3.5 ml of 10 mM sodium phosphate pH6.2, 1mM PMSF was applied and the eluant collected. F222 and DPDA oxidized F222 were precipitated with 9 volumes of acetone at -20°C. The samples were resuspended at 10 mg/ml in 8M urea in 88% formic acid and allowed to sit for 5 minutes. An equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein) and the samples incubated for 2 hours at room temperature in the dark. Prior to gel electrophoresis, the samples were lyophilized and resuspended at 2-5 mg/ml in sample buffer (1% pyridine, 5% NaDodSO₄, 5% glycerol and bromophenol blue) and disassociated at 95°C for 3 minutes. The samples were electrophoresed on discontinuous polyacrylamide gels (Kyte, J., et al. (1953) Anal. Bioch. 133 , 515-522). The gels were stained using the Pharmacia silver staining technique (Sammons, D.W., et al. (1981) Electrophoresis 2 135-141). The results of this experiment are shown in Figure 8. As can be seen, F222 treated with CNBr only gives nine resolved bands on SPG. However, when F222 is also treated with DPDA prior to cleavage, bands X, 7 and 9 disappear whereas bands 5 and 6 are greatly increased in intensity. In order to determine which of the methionines were effected, each of the CNBr peptides was isolated by reversed phase HPLC and further characterized. The buffer system in both Solvent A (aqueous) and Solvent B (organic) for all HPLC separations was 0.05% triethylamime/trifloroacetic acid (TEA-TFA). In all cases unless noted, solvent A consisted of 0.05% TEA-TFA in H₂0, solvent B was 0.05% TEA-TFA in 1-propanol, and the flow rate was 0.5 ml/minute. For HPLC analysis, two injections of 1 mg enzyme digest were used. Three samples were acetone precipitated, washed and dried. The dried 1 mg samples were resuspended at 10 mg/ml in 8M urea, 88% formic acid; an equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/ml protein). After incubation for 2 hours in the dark at room temperature, the samples were desalted on a 0.8 cm X 7 cm column of Tris Acryl GF05 coarse resin (IBF, Paris, France) equilibrated with 40% solvent B, 60% solvent A. 200 ul samples were applied at a flow rate of 1 ml a minute and 1.0-1.2 ml collected by monitoring the absorbance at 280nm. Prior to injection on the HPLC, each desalted sample was diluted with 3 volumes of solvent A. The samples were injected at 1.0 ml/min (2 minutes) and the flow then adjusted to 0.5 ml/min (100% A). After 2 minutes, a linear gradient to 60% B at 1.0% B/min was initiated. From each 1 mg run, the pooled peaks were sampled (50ul) and analyzed by gel electrophoresis as described above. Each polypeptide isolated by reversed phase HPLC was further analyzed for homogeneity by SPG. The position of each peptide on the known gene sequence (Wells, J.A., et al. (1983) Nucleic Acids Res. 11 7911-7924) was obtained through a combination of amino acid compositional analysis and, where needed, amino terminal sequencing. Prior to such analysis the following peptides were to rechromatographed. 1. CNBr peptides from F222 not treated with DPDA: Peptide 5 was subjected to two additional reversed phase separations. The 10 cm C4 column was equilibrated to 80%A/ 20%B and the pooled sample applied and washed for 2 minutes. Next an 0.5% ml B/min gradient was initiated. Fractions from this separation were again rerun, this time on the 25 cm C4 column, and employing 0.05% TEA-TFA in acetonitrile/1-propanol (1:1) for solvent B. The gradient was identical to the one just described. Peptide "X" was subjected to one additional separation after the initial chromatography. The sample was applied and washed for 2 minutes at 0.5ml/min (100%A), and a 0.5% ml B/min gradient was initiated. Peptides 7 and 9 were rechromatographed in a similar manner to the first rerun of peptide 5. Peptide 8 was purified to homogeneity after the initial separation. 2. CNBr Peptides from DPDA Oxidized F222: Peptides 5 and 6 from a CNBr digest of the oxidized F222 were purified in the same manner as peptide 5 from the untreated enzyme. Amino acid compositional analysis was obtained as follows. Samples (-1nM each amino acid) were dried, hydrolyzed in vacuo with 100 ul 6N HCl at 106°C for 24 hours and then dried in a Speed Vac. The samples were analyzed on a Beckmann 6300 AA analyzer employing ninhydrin detection. Amino terminal sequence data was obtained as previously described (Rodriguez, H., et al. (1984) Anal. Biochem. 134 , 538-547). The results are shown in Table VII and Figure 9. Amino and COOH terminii of CNBr fragments Terminus and Method Fragment amino, method COOH, method X 1, sequence 50, composition 9 51, sequence 119, composition 7 125, sequence 199, composition 8 200, sequence 275, composition 5ox 1, sequence 119, composition 6ox 120, composition 199, composition Peptides 5ox and 6ox refer to peptides 5 and 6 isolated from CNBr digests of the oxidized protein where their respective levels are enhanced. From the data in Table VII and the comparison of SPG tracks for the oxidized and native protein digests in Figure 8, it is apparent that (1) Met50 is oxidized leading to the loss of peptides X and 9 and the appearance of 5; and (2) Met124 is also oxidized leading to the loss of peptide 7 and the accumulation of peptide 6. Thus oxidation of B . amyloliquifaciens subtilisin with the peracid, diperdocecanoic acid leads to the specific oxidation of methionine at residues 50 and 124. EXAMPLE 2 Substitution at Met50 and Met124 in Subtilisin Met222Q The choice of amino acid for substitution at Met50 was based on the available sequence data for subtilisins from B . licheniformis (Smith, E.C., et al. (1968) J. Biol. Chem. 243 , 2184-2191), B.DY (Nedkov, P., et al. (1983) Hoppe Sayler's Z. Physiol. Chem. 364 1537-1540), B . amylosacchariticus (Markland, F.S., et al. (1967) J. Biol. Chem. 242 5198-5211) and B . subtilis (Stahl, M.L., et al. (1984) J. Bacteriol. 158 , 411-418). In all cases, position 50 is a phenylalanine. See Figure 5. Therefore, Phe50 was chosen for construction. At position 124, all known subtilisins possess a methionine. See Figure 5. Molecular modelling of the x-ray derived protein structure was therefore rehired to determine the most probable candidates for substitution. From all 19 candidates, isoleucine and leucine were chosen as the best residues to employ. In order to test whether or not modification at one site but not both was sufficient to increase oxidative stability, all possible combinations were built on the Q222 backbone (F50/Q222, I124/Q222, F50/I124/Q222). A. Construction of Mutations Between Codons 45 and 50 All manipulations for cassette mutagenesis were carried out on pS4.5 using methods disclosed in EPO Publication No. 0130756 and Wells, J.A., et al , (1985) Gene 34 , 315-323. The pΔ50 in Fig. 10, line 4, mutations was produced using the mutagenesis primer shown in Fig. 10, line 6, and employed an approach designated as restriction-purification which is described below. Briefly, a M13 template containing the subtilisin gene, M13mp11-SUBT was used for heteroduplex synthesis (Adelman, et al (1983), DNA 2 , 183-193). Following transfection of JM101 (ATCC 33876), the 1.5 kb Eco RI- Bam HI fragment containing the subtilisin gene was subcloned from M13mp11 SUBT rf into a recipient vector fragment of pBS42 the construction of which is described in EPO Publication No. 0130756. To enrich for the mutant sequence (pΔ50, line 4), the resulting plasmid pool was digested with Kpn I, and linear molecules were purified by polyacrylamide gel electrophoresis. Linear molecules were ligated back to a circular form, and transformed into E . coli MM294 cells (ATCC 31446). Isolated plasmids were screened by restriction analysis for the Kpn I, site. Kpn I⁺ plasmids were sequenced and confirmed the pΔ50 sequence. Asterisks in Figure 11 indicate the bases that are mutated from the wid type sequence (line 4). pΔ50 (line 4) was cut with Stu I and Eco RI and the 0.5 Kb fragment containing the 5' half of the subtilisin gene was purified (fragment 1). pΔ50 (line 4) was digested with Kpn I and Eco RI and the 4.0 Kb fragment containing the 3' half of the subtilisin gene and vector sequences was purified (fragment 2). Fragments 1 and 2 (line 5), and duplex DNA cassettes coding for mutations desired (shaded sequence, line 6) were mixed in a molar ratio of 1:1:10, respectively. For the particular construction of this example the DNA cassette contained the triplet TTT for codon 50 which encodes Phe. This plasmid was designated pF50. The mutant subtilisin was designated F50. B. Construction of Mutation Between Codons 122 and 127 The procedure of Example 2A was followed in substantial detail except that the mutagenesis primer of Figure 11, line 7 was used and restriction-purification for the Eco RV site in pΔ124 was used. In addition, the DNA cassette (shaded sequence, Figure 11, line 6) contained the triplet ATT for codon 124 which encodes Ile and CTT for Leu. Those plasmids which contained the substitution of Ile for Met124were designeated pI124. The mutant subtilisin was designated I124. C. Construction of Various F50/I124/Q222 Multiple Mutants The triple mutant, F50/I124/Q222, was constructed from a three-way ligation in which each fragment contained one of the three mutations. The single mutant Q222 (pQ222) was prepared by cassette mutagenesis as described in EPO Publication No. 0130756. The F50 mutation was contained on a 2.2kb Ava II to Pvu II fragment from pF50; the I124 mutation was contained on a 260 bp Pvu II to Ava II fragment from pI124; and the Q222 mutation was contained on 2.7 kb Ava II to Ava II fragment from pQ222. The three fragments were ligated together and transformed into E . coli MM294 cells. Restriction analysis of plasmids from isolated transformants confirmed the construction. To analyze the final construction it was convenient that the Ava II site at position 798 in the wild-type subtilisin gene was eliminated by the I124 construction. The F50/Q222 and I124/Q222 mutants were constructed in a similar manner except that the appropriate fragment from pS4.5 was used for the final construction. D. Oxidative Stability of Q222 Mutants The above mutants were analyzed for stability to peracid oxidation. As shown in Fig. 12, upon incubation with diperdodecanoic acid (protein 2mg/mL, oxidant 75ppm[0]), both the I124/Q222 and the F50/I124/Q222 are completely stable whereas the F50/Q222 and the Q222 are inactivated. This indicates that conversion of Met124 to I124 in subtilisin Q222 is sufficient to confer resistance to organic peracid oxidants. EXAMPLE 3 Subtilisin Mutants Having Altered Substrate Specificity-Hydrophobic Substitutions at Residues 166 Subtilisin contains an extended binding cleft which is hydrophobic in character. A conserved glycine at residue 166 was replaced with twelve non-ionic amino acids which can project their side-chains into the S-1 subsite. These mutants were constructed to determine the effect of changes in size and hydrophobicity on the binding of various substrates. A. Kinetics for Hydrolysis of Substrates Having Altered P-1 Amino Acids by Subtilisin from B. Amyloliquefaciens Wild-type subtilisin was purified from B . subtilis culture supernatants expressing the B . amyloliquefaciens subtilisin gene (Wells, J.A., et al . (1983) Nucleic Acids Res. 11 , 7911-7925) as previously described (Estell, D.A., et al . (1985) J. Biol. Chem. 260 , 6518-6521). Details of the synthesis of tetrapeptide substrates having the form succinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide (where X is the P1 amino acid) are described by DelMar, E.G., et al . (1979) Anal. Biochem. 99 , 316-320. Kinetic parameters, Km(M) and kcat(s⁻¹) were measured using a modified progress curve analysis (Estell, D.A., et al . (1985) J. Biol. Chem. 260 , 6518-6521). Briefly, plots of rate versus product concentration were fit to the differential form of the rate equation using a non-linear regression algorithm. Errors in kcat and Km for all values reported are less than five percent. The various substrates in Table VIII are ranged in order of decreasing hydrophobicity. Nozaki, Y. (1971), J. Biol. Chem. 246 , 2211-2217; Tanford C. (1978) Science 200 , 1012). P1 substrate Amino Acid kcat(S⁻¹) 1/Km(M⁻¹) kcat/Km (s-¹M-1) Phe 50 7,100 360,000 Tyr 28 40,000 1,100,000 Leu 24 3,100 75,000 Met 13 9,400 120,000 His 7.9 1,600 13,000 Ala 1.9 5,500 11,000 Gly 0.003 8,300 21 Gln 3.2 2,200 7,100 Ser 2.8 1,500 4,200 Glu 0.54 32 16 The ratio of kcat/Km (also referred to as catalytic efficienty) is the apparent second order rate constant for the conversion of free enzyme plus substrate (E+S) to enzyme plus products (E+P) (Jencks, W.P., Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287). The log (kcat/Km) is proportional to transition state binding energy, ΔG ≠ / T. A plot of the log kcat/Km versus the hydrophobicity of the P1 side-chain (Figure 14) shows a strong correlation (r = 0.98), with the exception of the glycine substrate which shows evidence for non-productive binding. These data show that relative differences between transition-state binding energies can be accounted for by differences in P-1 side-chain hydrophobicity. When the transition-state binding energies are calculated for these substrates and plotted versus their respective side-chain hydrophobicities, the line slope is 1.2 (not shown). A slope greater than unity, as is also the case for chymotrypsin (Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Harper, J.W., et al . (1984) Biochemistry , 23 , 2995-3002), suggests that the P1 binding cleft is more hydrophobic than ethanol or dioxane solvents that were used to empirically determine the hydrophobicity of amino acids (Nozaki, Y., et al . J. Biol. Chem. (1971) 246 , 2211-2217; Tanford, C. (1978) Science 200 , 1012). For amide hydrolysis by subtilisin, kcat can be interpreted as the acylation rate constant and Km as the dissociation constant, for the Michaelis complex (E·S), Ks. Gutfreund, H., et al (1956) Biochem. J. 63 , 656. The fact that the log kcat, as well as log 1/Km, correlates with substrate hydrophobicity is consistent with proposals (Robertus, J.D., et al . (1972) Biochemistry 11 , 2439-2449; Robertus, J.D., et al . (1972) Biochemistry 11 , 4293-4303) that during the acylation step the P-1 side-chain moves deeper into the hydrophobic cleft as the substrate advances from the Michaelis complex (E·S) to the tetrahedral transition-state complex (E·S ≠ ). However, these data can also be interpreted as the hydrophobicity of the P1 side-chain effecting the orientation, and thus the susceptibility of the scissile peptide bond to nucleophilic attack by the hydroxyl group of the catalytic Ser221. The dependence of kcat/Km on P-1 side chain hydrophobicity suggested that the kcat/Km for hydrophobic substrates may be increased by increasing the hydrophobicity of the S-1 binding subsite. To test this hypothesis, hydrophobic amino acid substitutions of Gly166 were produced. Since hydrophobicity of aliphatic side-chains is directly proportional to side-chain surface area (Rose, G.D., et al . (1985) Science 229 , 834-838; Reynolds, J.A., et al . (1974) Proc. Natl. Acad. Sci. USA 71 , 2825-2927), increasing the hydrophobicity in the S-1 subsite may also sterically hinder binding of larger substrates. Because of difficulties in predicting the relative importance of these two opposing effects, we elected to generate twelve non-charged mutations at position 166 to determine the resulting specificities against non-charged substrates of varied size and hydrophobicity. B. Cassette Mutagenesis of the P1 Binding Cleft The preparation of mutant subtilisims containing the substitution of the hydrophobic amino acids Ala, Val and Phe into residue 166 has been described in EPO Publication No. 0130756. The same method was used to produce the remaining hydrophobic mutants at residue 166. In applying this method, two unique and silent restriction sites were introduced in the subtilisin genes to closely flank the target codon 166. As can be seen in Figure 13, the wild type sequence (line 1) was altered by site-directed mutagenesis in M13 using the indicated 37mer mutagenesis primer, to introduce a 13 bp delection (dashedline) and unique Sac I and Xma I sites (underlined sequences) that closely flank codon 166. The subtilisin gene fragment was subcloned back into the E . coli - B . subtilis shuttle plasmid, pBS42, giving the plasmid pΔ166 (Figure 13, line 2). pΔ166 was cut open with Sac I and Xma I, and gapped linear molecules were purified (Figure 13, line 3). Pools of synthetic oligonucleotides containing the mutation of interest were annealed to give duplex DNA cassettes that were ligated into gapped pΔ166 (underlined and overlined sequences in Figure 13, line 4). This construction restored the coding sequence except over position 166(NNN; line 4). Mutant sequences were confirmed by dideoxy sequencing. Asterisks denote sequence changes from the wild type sequence. Plasmids containing each mutant B . amyloliquefaciens subtilisin gene were expressed at roughly equivalent levels in a protease deficient strain of B . subtilis , BG2036 as previously described. EPO Publication No. 0130756; Yang, M., et al . (1984) J. Bacteriol. 160 , 15-21; Estell, D.A., et al (1985) J. Biol. Chem. 260 , 6518-6521. C. Narrowing Substrate Specificity by Steric Hindrance To probe the change in substrate specificity caused by steric alterations in the S-1 subsite, position 166 mutants were kinetically analyzed versus P1 substrates of increasing size (i.e., Ala, Met, Phe and Tyr). Ratios of kcat/Km are presented in log form in Figure 15 to allow direct comparisons of transition-state binding energies between various enzyme-substrate pairs. According to transition state theory, the free enery difference between the free enzyme plus substrate (E + S) and the transition state complex (E·S ≠ ) can be calculated from equation (1), in which kcat is the turnover number, Km is the Michaelis constant, R is the gas constant, T is the temperature, k is Boltzmann's constant, and h is Planck's constant. Specificity differences are ezpressed quantitatively as differences between transition state binding energies (i.e., ΔΔG ≠ / t), and can be calculated from equation (2). A and B represent either two different substrates assayed againt the same enzyme, or two mutant enzymes assayed against the same substrate. As can be seen from Figure 15A, as the size of the side-chain at position 166 increases the substrate preference shifts from large to small P-1 side-chains. Enlarging the side-chain at position 166 causes kcat/Km to decrease in proportion to the size of the P-1 substrate side-chain (e.g., from Gly166 (wild-type) through W166, the kcat/Km for the Tyr substrate is decreased most followed in order by the Phe, Met and Ala P-1 substrates). Specific steric changes in the position 166 side-chain, such as he presence of a β-hydroxyl group, β- or γ-aliphatic branching, cause large decreases in kcat/Km for larger P1 substrates. Introducing a β-hydroxyl group in going from A166 (Figure 15A) to S166 (Figure 15B), causes an 8 fold and 4 fold reduction in kcat/Km for Phe and Tyr substrates, respectively, while the values for Ala and Met substrates are unchanged. Producing a β-branched structure, in going from S166 to T166, results in a drop of 14 and 4 fold in kcat/Km for Phe and Tyr, respectively. These differences are slightly magnified for V166 which is slightly larger and isosteric with T166. Enlarging the β-branched substituents from V166 to I166 causes a lowering of kcat/Km between two and six fold toward Met, Phe and Tyr substrates. Inserting a γ-branched structure, by replacing M166 (Figure 15A) with L166 (Figure 15B), produces a 5 fold and 18 fold decrease in kcat/Km for Phe and Tyr substrates, respectively. Aliphatic γ-branched appears to induce less steric hindrance toward the Phe P-1 substrate than β-branching, as evidenced by the 100 fold decrease in kcat/Km for the Phe substrate in going from L166 to I166. Reductions in kcat/Km resulting from increases in side chain size in the S-1 subsite, or specific structural features such as β- and γ-branching, are quantitatively illustrated in Figure 16. The kcat/Km values for the position 166 mutants determined for the Ala, Met, Phe, and Tyr P-1 substrates (top panel through bottom panel, respectively), are plotted versus the position 166 side-chain volumes (Chothia, C. (1984) Ann. Rev. Biochem. 53 , 537-572). Catalytic efficiency for the Ala substrate reaches a maximum for I166, and for the Met substrate it reaches a maximum between V166 and L166. The Phe substrate shows a broad kcat/Km peak but is optimal with A166. Here, the β-branched position 166 substitutions form a line that is parallel to, but roughly 50 fold lower in kcat/Km than side-chains of similar size [i.e., C166 versus T166, L166 versus I166). The Tyr substrate is most efficiently utilized by wild type enzyme (Gly166), and there is a steady decrease as one proceeds to large position 166 side-chains. The β-branched and γ-branched substitutions form a parallel line below the other non-charged substitutions of similar molecular volume. The optimal substitution at position 166 decreases in volume with increasing volume of the P1 substrate [i.e., I166/Ala substrate, L166/Met substrate, A166/Phe substrate, Gly166/Tyr substrate]. The combined volumes for these optimal pairs may approximate the volume for productive binding in the S-1 subsite. For the optimal pairs, Gly166/Tyr substrate, A166/Phe substrate, L166/Met substrate, V166/Met substrate, and I166/Ala substrate, the combined volumes are 266,295,313,339 and 261 A³, respectively. Subtracting the volume of the peptide backbone from each pair (i.e., two times the volume of glycine), an average side-chain volume of 160±32A³ for productive binding can be calculated. The effect of volume, in excess to the productive binding volume, on the drop in transition-state binding energy can be estimated from the Tyr substrate curve (bottom panel, Figure 16), because these data, and modeling studies (Figure 2), suggest that any substitution beyond glycine causes steric repulsion. A best-fit line drawn to all the data (r = 0.87) gives a slope indicating a loss of roughly 3 kcal/mol in transition state binding energy per 100A³ of excess volume. (100A³ is approximately the size of a leucyl side-chain.) D. Enhanced Catalytic Efficiency Correlates with Increasing Hydrophobicity of the Position 166 Substitution Substantial increases in kcat/Km occur with enlargement of the position 166 side-chain, except for the Tyr P-1 substrate (Figure 16). For example, kcat/Km increases in progressing from Gly166 to I166 for the Ala substrate (net of ten-fold), from Gly166 to L166 for the Met substrate (net of ten-fold) and from Gly166 to A166 for the Phe substrate (net of two-fold). The increases in kcat/Km cannot be entirely explained by the attractive terms in the van der Waals potential energy function because of their strong distance dependence (1/r⁶) and because of the weak nature of these attractive forces (Jencks, W.P., Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Levitt, M. (1976) J. Mol. Biol. 104 , 59-107). For example, Levitt (Levitt, M. (1976) J. Mol. Biol. 104 , 59-107) has calculated that the van der Waals attraction between two methionyl residues would produce a maximal interaction energy of roughly -0.2 kcal/mol. This energy would translate to only 1.4 fold increase in kcat/Km. The increases of catalytic efficiency caused by side-chain substitutions at position 166 are better accounted for by increases in the hydrophobicity of the S-1 subsite. The increase kcat/Km observed for the Ala and Met substrates with increasing position 166 side-chain size would be expected, because hydrophobicity is roughly proportional to side-chain surface area (Rose, G.D., et al . (1985) Science 229 , 834-838; Reynolds, J.A., et al . (1974) Proc. Natl. Acad. Sci. USA 71 , 2825-2927). Another example that can be interpreted as a hydrophobic effect is seen when comparing kcat/Km for isosteric substitutions that differ in hydrophobicity such as S166 and C166 (Figure 16). Cysteine is considerably more hydrophobic than serine (-1.0 versus +0.3 kcal/mol) (Nozaki, Y., et al . (1971) J. Biol. Chem. 246 , 2211-2217; Tanford, C. (1978) Science 200 , 1012). The difference in hydrophobicity correlates with the observation that C166 becomes more efficient relative to Ser166 as the hydrophobicity of the substrates increases (i.e., Ala < Met < Tye < Phe). Steric hindrance cannot explain these differences because serine is considerably smaller than cysteine (99 versus 118A³). Paul, I.C., Chemistry of the -SH Group (ed. S. Patai, Wiley Interscience, New York, 1974) pp. 111-149. E. Production of an Elastase-Like Specificity in Subtilisin The I166 mutation illustrates particularly well that large changes in specificity can be produced by altering the structure and hydrophobicity of the S-1 subsite by a single mutation (Figure 17). Progressing through the small hydrophobic substrates, a maximal specificity improvement over wild type occurs for the Val substrate (16 fold in kcat/Km). As the substrate side chain size increases, these enhancements shrink to near unity (i.e., Leu and His substrates). The I166 enzyme becomes poorer against larger aromatic substrates of increasing size (e.g., I166 is over 1,000 fold worse against the Tyr substrate than is Gly166). We interpret the increase in catalytic efficiency toward the small hydrophobic substrates for I166 compared to Gly166 to the greater hydrophobicity of isoluecine (i.e., -1.8 kcal/mol versus 0). Nozaki, Y., et al . (1971) J. Biol. Chem. 246 , 2211-2217; Tanford, C. (1978) Science 200 , 1012. The decrease in catalytic efficiency toward the very large substrates for I166 versus Gly166 is attributed to steric repulsion. The specificity differences between Gly166 and I166 are similar to the specificity differences between chymotrypsin and the evolutionary relative, elastase (Harper, J.W., et al (1984) Biochemistry 23 , 2995-3002). In elastase, the bulky amino acids, Thr and Val, block access to the P-1 binding site for large hydrophobic substrates that are preferred by chymotrypsin. In addition, the catalytic efficiencies toward small hydrophobic substrates are greater for elastase than for chymotrypsin as we obeseve for I166 versus Gly166 in subtilisin. EXAMPLE 4 Substitution of Ionic Amino Acids for Gly166 The construction of subtilisin mutants containing the substitution of the ionic amino acids Asp, Asn, Gln, Lys and Ang are disclosed in EPO Publication No. 0130756. The present example describes the construction of the mutant subtilisin containing Glu at position 166 (E166) and presents substrate specificity data on these mutants. Further data on position 166 and 156 single and double mutants is presented infra . pΔ166, described in Example 3, was digested with SacI and XmaI. The double strand DNA cassette (underlined and overlined) of line 4 in Figure 13 contained the triplet GAA for the codon 166 to encode the replacement of Glu for Gly166. This mutant plasmid designated pQ166 was propagated in BG2036 as described. This mutant subtilisin, together with the other mutants containing ionic substituent amino acids at residue 166, were isolated as described and further analyzed for variations in substrate specificity. Each of these mutants was analyzed with the tetrapeptide substrates, succinyl-L-AlaL-AlaProL-X-p-nitroanilide, where X was Phe, Ala and Glu. The results of this analysis are shown in Table IX. Position 166 P-1 Substrate (kcat/Km x 10⁻⁴) Phe Ala Glu Gly (wild type) 36.0 1.4 0.002 Asp (D) 0.5 0.4 <0.001 Glu (E) 3.5 0.4 <0.001 Asn (N) 18.0 1.2 0.004 Gln (Q) 57.0 2.6 0.002 Lys (K) 52.0 2.8 1.2 Arg (R) 42.0 5.0 0.08 These results indicate that charged amino acid substitutions at Gly166 have improved catalytic efficiencies (kcat/Km) for oppositely charged P-1 substrates (as much as 500 fold) and poorer catalytic efficiency for like charged P-1 substrates. EXAMPLE 5 Substitution of Glycine at Position 169 The substitution of Gly169 in B . amyloliquefaciens subtilisin with Ala and Ser is described in EPO Publication No. 0130756. The same method was used to make the remaining 17 mutants containing all other substituent amino acids for position 169. The construction protocol is summarized in Figure 18. The overscored and underscored double stranded DNA cassettes used contained the following triplet encoding the substitution of the indicated amino acid at residue 169. GCT A ATG M TGT C AAC N GAT D CCT P GAA E CAA Q TTC F AGA R GGC G AGC S CAC H ACA T ATC I GTT V AAA K TGG W CTT L TAC Y Each of the plasmids containing a substituted Gly169 was designated pX169, where X represents the substituent amino acid. The mutant subtilisins were simialrly designated. Two of the above mutant subtilisins, A169 and S169, were analyzed for substrate specificity against synthetic substrates containing Phe, Leu, Ala and Arg in the P-1 position. The following results are shown in Table X. Effect of Serine and Alanine Mutations at Position 169 on P-1 Substrate Specificity Position 169 P-1 Substrate [kcat/Km x 10⁻⁴) Phe Leu Ala Arg Gly (wild type) 40 10 1 0.4 A169 120 20 1 0.9 S169 50 10 1 0.6 These results indicate that substitutions of Ala and Ser at Gly169 have remarkably similar catalytic efficiencies against a range of P-1 substrates compared to their position 166 counterparts. This is probably because position 169 is at the bottom of the P-1 specificity subsite. EXAMPLE 6 Substitution at Position 104 Tyr104 has been substituted with Ala, His, Leu, Met and Ser. The method used was a modification of the site directed mutagenesis method. According to the protocol of Figure 19, a primer (shaded in line 4) introduced a unique Hind III site and a frame shift mutation at codon 104. Restriction-purification for the unique Hind III site facilitated the isolation of the mutant sequence (line 4). Restriction-selection against this Hind III site using pimers in line 5 was used to obtain position 104 mutants. The following triplets were used in the primers of Figure 19, line 5 for the 104 codon which substituted the following amino acids. GCT A TTC F ATG M CCT P CTT L ACA T AGC S TGG W CAC H TAC Y CAA Q GTT V GAA E AGA R GGC G AAC N ATC I GAT D AAA K TGT C The substrates in Table XI were used to analyze the substrate specificity of these mutants. The results obtained fo H104 subtilisin are shown in Table XI. Substrate kcat Km Kcat/Km WT H104 WT H104 WT H104 sAAPFpNA 50.0 22.0 1.4x10⁻⁴ 7.1x10⁻⁴ 3.6x10⁵ 3.1x10⁴ sAAPApNA 3.2 2.0 2.3x10⁻⁴ 1.9x10⁻³ 1.4x10⁴ 1x10³ sFAPFpNA 26.0 38.0 1.8x10⁻⁴ 4.1x10⁻⁴ 1.5x10⁵ 9.1x10⁴ sFAPApNA 0.32 2.4 7.3x10⁻⁵ 1.5x10⁻⁴ 4.4x10³ 1.6x10⁴ From these data it is clear that the substitution of His for Tyr at position 104 produces an enzyme which is more efficient (higher kcat/Km) when Phe is at the P-4 substrate position than when Ala is at the P-4 substrate position. EXAMPLE 7 Substitution of Ala152 Ala152 has been substituted by Gly and Ser to determine the effect of such substitutions on substrate specificity. The wild type DNA sequence was mutated by the V152/P153 primer (Figure 20, line 4) using the above restriction-purification approach for the new Kpn I site. Other mutant primers (shaded sequences Figure 20; S152, line 5 and G152, line 6) mutated the new Kpn I site away and such mutants were isolated using the restriction-selection procedure as described above for loss of the Kpn I site. The results of these substitutions for the above synthetic substrates containing the P-1 amino acids Phe, Leu and Ala are shown in Table XII. Position 152 P-1 Substrate (kcat/Kmx10⁻⁴) Phe Leu Ala Gly (G) 0.2 0.4 <0.04 Ala (wild type) 40.0 10.0 1.0 Ser (S) 1.0 0.5 0.2 These results indicate that, in contrast to positions 166 and 169, replacement of Ala152 with Ser or Gly causes a dramatic reduction in catalytic efficiencies across all substrates tested. This suggests Ala152, at the top of the S-1 subsite, may be the optimal amino acid because Ser end Gly ore homologous Ala substitutes. EXAMPLE 8 Substitution at Position 156 Mutants containing the substitution of Ser and Gln for Glu156 have been constructed according to the overall method depicted in Figure 21. This method was designed to facilitate the construciton of multiple mutants at position 156 and 166 as will be described hereinafter. However, by regenerating the wild type Gly166, single mutations at Glu156 were obtained. The plasmid pΔ166 is already depicted in line 2 of Figure 13. The synthetic oligonucleotides at the top right of Figure 21 represent the same DNA cassettes depicted in line 4 of Figure 13. The plasmid p166 in Figure 21 thus represents the mutant plasmids of Examples 3 and 4. In this particular example, p166 contains the wild type Gly166. Construction of position 156 single mutants were prepared by ligation of the three fragments (1-3) indicated at the bottom of Figure 21. Fragment 3, containing the carboxy-terminal portion of the subtilisin gene including the wild type position 166 codon, was isolated as a 610 bp Sac I- Bam HI fragment. Fragment 1 contained the vector sequences, as well as the amino-terminal sequences of the subtilisin gene through codon 151. To produce fragment 1, a unique Kpn I site at codon 152 was introduced into the wild type subtilisin sequence from pS4.5. Site-directed mutagenesis in M13 employed a primer having the sequence 5'-TA-GTC-GTT-GCG-GTA-CCC-GGT-AAC-GAA-3' to produce the mutation. Enrichment for the mutant sequence was accomplished by restriction with Kpn I, purification and self ligation. The mutant sequence containing the Kpn I site was confirmed by direct plasmid sequencing to give pV152. pV152 (∼1 µg) was digested with Kpn I and treated with 2 units of DNA polymerase I large fragment (Klenow fragment from Boeringer-Mannheim) plus 50 µM deoxynucleotide triphosphates at 37°C for 30 min. This created a blunt end that terminated with codon 151. The DNA was extracted with 1:1 volumes phenol and CHCl₃ and DNA in the aqueous phase was precipitated by addition of 0.1 volumes 5M ammonium acetate and two volumes ethanol. After centrifugation and washing the DNA pellet with 70% ethanol, the DNA was lyophilized. DNA was digested with BamHI and the 4.6kb piece (fragment 1) was purified by acrylamide gel electrophoresis followed by electroelution. Fragment 2 was a duplex synthetic DNA cassette which when ligated with fragments 1 and 3 properly restored the coding sequence except at codon 156. The top strand was synthesized to contain a glutamine codon, and the complementary bottom strand coded for serine at 156. Ligation of heterophosphorylated cassettes leads to a large and favorable bias for the phosphorylated over the non-phosphorylated oligonucleotide sequence in the final segrated plasmid product. Therefore, to obtain Q156 the top strand was phosphorylated, and annealed to the non-phosphorylated bottom strand prior to ligation. Similarly, to obtain S156 the bottom strand was phosphorylated and annealed to the non-phosphorylated top strand. Mutant sequences were isolated after ligation and transformation, and were confirmed by restriction analysis and DNA sequencing as before. To express variant subtilisins, plasmids were transformed into a subtilisin-neutral protease deletion mutant of B . subtilis , BG2036, as previously described. Cultures were fermented in shake flasks for 24 h at 37°C in LB media containing 12.5 mg/mL chloramphenicol and subtilisin was purified from culture supernatants as described. Purity of subtilisin was greater than 95% as judged by SDS PAGE. These mutant plasmids designated pS156 and pQ156 and mutant subtilisins designated S156 and Q156 were analyzed with the above synthetic substrates where P-1 comprised the amino acids Glu, Gln, Met and Lys. The results of this analyses are presented in Example 9. EXAMPLE 9 Multiple Mutants With Altered Substrate Specificity - Substitution at Positions 156 and 166 Single substitutions of position 166 are described in Examples 3 and 4. Example 8 describes single substitutions at position 156 as well as the protocol of Figure 21 whereby various double mutants comprising the substitution of various amino acids at positions 156 and 166 can be made. This example describes the construction and substrate specificity of subtilisin containing substitutions at position 156 and 166 and summarizes some of the data for single and double mutants at positions 156 and 166 with various substrates. K166 is a common replacement amino acid in the 156/166 mutants described herein. The replacement of Lys for Gly166 was achieved by using the synthetic DNA cassette at the top right of Figure 21 which contained the triplet AAA for NNN. This produced fragment 2 with Lys substituting for Gly166. The 156 substituents were Gln and Ser. The Gln and Ser substitutions at Gly156 are contained within fragment 3 (bottom right Figure 21). The multiple mutants were produced by combining fragments 1, 2 and 3 as described in Example 8. The mutants Q156/K166 and S156/K166 were selectively generated by differential phosphorylation as described. Alternatively, the double 156/166 mutants, c.f. Q156/K166 and S156/K166, were prepared by ligation of the 4.6kb Sac I- Bam HI fragment from the relevant p156 plasmid containing the 0.6kb Sac I- Bam HI fragment from the relevant p166 plasmid. These mutants, the single mutant K166, and the S156 and Q156 mutants of Example 8 were analyzed for substitute specificity against synthetic polypeptides containing Phe or Glu as the P-1 substrate residue. The results are presented in Table XIII. As can be seen in Table XIV, either of these single mutations improve enzyme performance upon substrates with glutamate at the P-1 enzyme binding site. When these single mutations were combined, the resulting multiple enzyme mutants are better than either parent. These single or multiple mutations also alter the relative pH activity profiles of the enzymes as shown in Figure 23. To isolate the contribution of electrostatics to substrate specificity from other chemical binding forces, these various single and double mutants were analyzed for their ability to bind and cleave synthetic substrates containing Glu, Gln, Met and Lys as the P-1 substrate amino acid. This permitted comparisons between side-chains that were more sterically similar but differed in charge (e.g., Glu versus Gln, Lys versus Met). Similarly, mutant enzymes were assayed against homologous P-1 substrates that were most sterically similar but differed in charge (Table XIV). The kcat/Km ratios shown are the second order rate constants for the conversion of substrate to product, and represent the catalytic efficiency of the enzyme. These ratios are presented in logarithmic form to scale the data, and because log kcat/Km is proportional to the lowering of transition-state activation energy (ΔG T ). Mutations at position 156 and 166 produce changes in catalytic efficiency toward Glu, Gln, Met and Lys P-1 substrates of 3100, 60, 200 and 20 fold, respectively. Making the P-1 binding-site more positively charged [e.g., compare Gln156/Lys166 (Q156/K166) versus Glu156/Met166 (Glu156/M166)] dramatically increased kcat/Km toward the Glu P-1 substrate (up to 3100 fold), and decreased the catalytic efficiency toward the Lys P-1 substrate (up to 10 fold). In addition, the results show that the catalytic efficiency of wild type enzyme can be greatly improved toward any of the four P-1 substrates by mutagenesis of the P-1 binding site. The changes in kcat/Km ore caused predominantly by changes in 1/Km. Because 1/Km is approximately equal to 1/Ks, the enzyme-substrate association constant, the mutations primarily cause a change in substrate binding. These mutations produce smaller effects on kcat that run parallel to the effects on 1/Km. The changes in kcat suggest either an alteration in binding in the P-1 binding site in going from the Michaelis-complex E·S) to the transition-state complex (E-S≠) as previously proposed (Robertus, J.D., et al . (1972) Biochemistry 11 , 2439-2449; Robertus, J.D., et al . (1972) Biochemistry 11 , 4293-4303), or change in the position of the scissile peptide bond over the catalytic serine in the E·S complex. Changes in substrate preference that arise from changes in the net charge in the P-1 binding site show trends that are best accounted for by electrostatic effects (Figure 28). As the P-1 binding cleft becomes more positively charged, the average catalytic efficiency increases much more for the Glu P-1 substrate than for its neutral and isosteric P-1 homolog, Gln (Figure 28A). Furthermore, at the positive extreme both substrates have nearly identical catalytic efficiencies. In contrast, as the P-1 site becomes more positively charged the catalytic efficiency toward the Lys P-1 substrate decreases, and diverges sharply from its neutral and isosteric homolog, Met (Figure 28B). The similar and parallel upward trend seen with increasing positive charge for the Met and Glu P-1 substrates probably results from the fact that all the substrates are succinylated on their amino-terminal end, and thus carry a formal negative charge. The trends observed in log kcat/Km are dominated by changes in the Km term (Figures 28C and 28D). As the pocket becomes more positively charged, the log 1/Km values converge for Glu and Gln P-1 substrates (Figure 28C), and diverge for Lys and Met P-1 substrates (Figure 28D). Although less pronounced effects are seen in log kcat, the effects of P-1 charge on log kcat parallel those seen in log 1/Km and become larger as the P-1 pocket becomes more positively charged. This may result from the fact that the transition-state is a tetrahedral anion, and a net positive charge in the enzyme may serve to provide some added stabilization to the transition-state. The effect of the change in P-1 binding-site charge on substrate preference can be estimated from the differences in slopes between the charged and neutral isosteric P-1 substrates (Figure 28B). The average change in substrate preference (Δlog kcat/Km) between charged and neutral isosteric substrates increases roughly 10-fold as the complementary charge or the enzyme increases (Table XV). When comparing Glu versus Lys, this difference is 100-fold and the change in substrate preference appears predominantly in the Km term. Differential Effect on Binding Site Charge on log kcat/Km or (log 1/Km) for P-1 Substrates that Differ in Charge Change in P-1 Binding Site Charge Δlog kcat/Km (Δlog 1/Km) GluGln MetLys GluLys -2 to -1 n.d. 1.2 (1.2) n.d. -1 to 0 0.7 (0.6) 1.3 (0.8) 2.1 (1.4) 0 to +1 1.5 (1.3) 0.5 (0.3) 2.0 (1.5) Avg. change in log kcat/K m or (log 1/Km) per unit charge change 1.1 (1.0) 1.0 (0.8) 2.1 (1.5) The free energy of electrostatic interactions in the structure and energetics of salt-bridge formation depends on the distance between the charges and the microscopic dielectric of the media. To dissect these structural and microenvironmental effects, the energies involved in specific salt-bridges were evaluated. In addition to the possible salt-bridges shown (Figures 29A and 29B), reasonable salt-bridges can be built between a Lys P-1 substrate and Asp at position 166, and between a Glu P-1 substrate and a Lys at position 166 (not shown). Although only one of these structures is confirmed by X-ray crystalography (Poulos, T.L., et al . (1976) J. Mol. Biol. 257 1097-1103), all models have favorable torsion angles (Sielecki, A.R., et al . (1979) J. Mol. Biol. 134 , 781-804), and do not introduce unfavorable van der Waals contacts. The change in charged P-1 substrate preference brought about by formation of the model salt-bridges above are shown in Table XVI. The difference between catalytic efficiencies (i.e., Δlog kcat/Km) for the charged and neutral P-1 substrates (e.g., Lys minus Met or Glu minus Gln) give the substrate preference for each enzyme. The change in substrate preference (ΔΔlog kcat/Km) between the charged and more neutral enzyme homologs (e.g., Glu156/Gly166 minus Gln156(Q156)/Gly166) reflects the change in catalytic efficiency that may be attributed solely to electrostatic effects. These results show that the average change in substrate preference is considerably greater when electrostatic substitutions are produced at position 166 (50-fold in kcat/Km) versus position 156 (12-fold in kcat/Km). From these ΔΔlog kcat/Km values, an average change in transition-state stabilization energy can be calculated of -1.5 and -2.4 kcal/mol for substitutions at positions 156 and 166, respectively. This should represent the stabilization energy contributed from a favorable electrostatic interaction for the binding of free enzyme and substrate to form the transition-state complex. EXAMPLE 10 Substitutions at Position 217 Tyr217 has been substituted by all other 19 amino acids. Cassette mutagenesis as described in EPO publication No. 0130756 was used according to the protocol of Figure 22. The Eco RV restriction site was used for restriction-purification of pΔ217. Since this position is involved in substrate binding, mutations here effect kinetic parameters of the enzyme. An example is the substitution of Leu for Tyr at position 217. For the substrate sAAPFpNa, this mutant has a kcat of 277 5' and a Km of 4.7x10⁻⁴ with a kcat/Km ratio of 6x10⁵. This represents a 5.5-fold increase in kcat with a 3-fold increase in Km over the wild type enzyme. In addition, replacement of Tyr217 by Lys, Arg, Phe or Leu results in mutant enzymes which are more stable at pHs of about 9-11 than the WT enzyme. Conversely, replacement of Tyr217 by Asp, Glu, Gly or Pro results in enzymes which are less stable at pHs of about 9-11 than the WT enzyme. EXAMPLE 11 Multiple Mutants Having Altered Thermal Stability B . amyloliquefacien subtilisin does not contain any cysteine residues. Thus, any attempt to produce thermal stability by Cys cross-linkage required the substitution of more than one amino acid in subtilisin with Cys. The following subtilisin residues were multiply substituted with cysteine: Thr22/Ser87 Ser24/Ser87 Mutagenesis of Ser24 to Cys was carried out with a 5' phosphorylated oligonucleotide primer having the sequence (Asterisks show the location of mismatches and the underlined sequence shows the position of the altered Sau 3A site.) The B . amyloliquefaciens subtilisin gene on a 1.5 kb Eco RI- BAM HI fragment from pS4.5 was cloned into M13mp11 and single stranded DNA was isolated. This template (M13mp11SUBT) was double primed with the 5' phosphorylated M13 universal sequencing primer and the mutagenesis primer. Adelman, et al . (1983) DNA 2 , 183-193. The heteroduplex was transfected into competent JM101 cells and plaques were probed for the mutant sequence (Zoller, M.J., et al . (1982) Nucleic Acid Res. 10 , 6487-6500; Wallace, et al . (1981) Nucleic Acid Res. 9 , 3647-3656) using a tetramethylammonium chloride hybridization protocol (Wood, et al . (1985) Proc. Natl. Acad. Sci. USA 82 , 1585-1588). The Ser87 to Cys mutation was prepared in a similar fashion using a 5' phosphorylated primer having the sequence (The asterisk indicates the position of the mismatch and the underlined sequence shows the position of a new Mst I site.) The C24 and C87 mutations were obtained at a frequency of one and two percent, respectively. Mutant sequences were confirmed by dideoxy sequencing in M13. Mutagenesis of Tyr21/Thr22 to A21/C22 was carried out with a 5' phosphorylated oligonucleotide primer having the sequence (The asterisks show mismatches to the wild type sequence and the underlined sequence shows the position of an altered Sau 3A site.) Manipulations for heteroduplex synthesis were identical to those described for C24. Because direct cloning of the heteroduplex DNA fragment can yield increased frequencies of mutagenesis, the Eco RI- Bam HI subtilisin fragment was purified and ligated into pBS42. E . coli MM 294 cells were transformed with the ligation mixture and plasmid DNA was purified from isolated transformants. Plasmid DNA was screened for the loss of the Sau 3A site at codon 23 that was eliminated by the mutagenesis primer. Two out of 16 plasmid preparations had lost the wild type Sau 3A site. The mutant sequence was confirmed by dideoxy sequencing in M13. Double mutants, C22/C87 and C24/C87, were constructed by ligating fragments sharing a common Cla I site that separated the single parent cystine codons. Specifically, the 500 bp Eco RI- Cla I fragment containing the 5' portion of the subtilisin gene (including codons 22 and 24) was ligated with the 4.7 kb Cla I- Eco RI fragment that contained the 3' portion of the subtilisin gene (including codon 87) plus pBS42 vector sequence. E . coli MM 294 was transformed with ligation mixtures and plasmid DNA was purified from individual transformants. Double-cysteine plasmid constructions were identified by restriction site markers originating from the parent cysteine mutants (i.e., C22 and C24, Sau 3A minus; Cys87, Mst I plus). Plasmids from E . coli were transformed into B . subtilis BG2036. The thermal stability of these mutants as compared to wild type subtilisin are presented in Figure 30 and Tables XVII and XVIII. Effect of DTT on the Half-Time of Autolytic Inactivation of Wild-Type and Disulfide Mutants of Subtilisin Enzyme t ½ -DTT/+DTT -DDT +DTT min Wild-type 95 85 1.1 C22/C87 44 25 1.8 C24/C87 92 62 1.5 Effect of Mutations in Subtilisin on the Half-Time of Autolytic Inactivation at 58°C Enzyme t ½ min Wild-type 120 C22 22 C24 120 C87 104 C22/C87 43 C24/C87 115 The disulfides introduced into subtilisin did not improve the autolytic stability of the mutant enzymes when compared to the wild-type enzyme. However, the disulfide bonds did provide a margin of autolytic stability when compared to their corresponding reduced double-cysteine enzyme. Inspection of a highly refined x-ray structure of wild-type B . amyloliquefaciens subtilisin reveals a hydrogen bond between Thr22 and Ser87. Because cysteine is a poor hydrogen donor or acceptor (Paul, I.C. (1974) in Chemistry of the -SH Group (Patai, S., ed.) pp. 111-149, Wiley Interscience, New York) weakening of 22/87 hydrogen bond may explain why the C22 and C87 single-cysteine mutant proteins are less autolytically stable than either C24 or wild-type (Table XVIII). The fact that C22 is less autolytically stable than C87 may be the result of the Tyr21A mutation (Table XVIII). Indeed, construction and analysis of Tyr21/C22 shows the mutant protein has an autolytic stability closer to that of C87. In summary, the C22 and C87 of single-cysteine mutations destabilize the protein toward autolysis, and disulfide bond formation increases the stability to a level less than or equal to that of wild-type enzyme. EXAMPLE 12 Multiple Mutants Containing Substitutions at Position 222 and Position 166 or 169 Double mutants 166/222 and 169/222 were prepared by ligating together (1) the 2.3kb Aca II fragment from pS4.5 which contains the 5' portion of the subtilisin gene and vector sequences, (2) the 200bp Ava II fragment which contains the relevant 166 or 169 mutations from the respective 166 or 169 plasmids, and (3) the 2.2kb Ava II fragment which contains the relevant 222 mutation 3' and of the subtilisin genes and vector sequence from the respective p222 plasmid. Although mutations at position 222 improve oxidation stability they also tend to increase the Km. An example is shown in Table XIX. In this case the A222 mutation was combined with the K166 mutation to give an enzyme with kcat and Km intermediate between the two parent enzymes. kcat Km WT 50 1.4x10⁻⁴ A222 42 9.9x10⁻⁴ K166 21 3.7x10⁻⁵ K166/A222 29 2.0x10⁻⁴ substrate sAAPFpNa EXAMPLE 13 Multiple Mutants Containing Substitutions at Positions 50, 156, 166, 217 and Combinations Thereof The double mutant S156/A169 was prepared by ligation of two fragments, each containing one of the relevant mutations. The plasmid pS156 was cut with Xma I and treated with S1 nuclease to create a blunt end at codon 167. After removal of the nuclease by phenol/chloroform extraction and ethanol precipitation, the DNA was digested with Bam HI and the approximately 4kb fragment containing the vector plus the 5' portion of the subtilisin gene through codon 167 was purified. The pA169 plasmid was digested with Kpn I and treated with DNA polymerase Klenow fragment plus 50 µM dNTPs to create a blunt end codon at codon 168. The Klenow was removed by phenol/chloroform extraction and ethanol precipitation. The DNA was digested with Bam HI and the 590bp fragment including codon 168 through the carboxy terminus of the subtilisin gene was isolated. The two fragments were then ligated to give S156/A169. Triple and quadruple mutants were prepared by ligating together (1) the 220bp Pvu II/ Hae II fragment containing the relevant 156, 166 and/or 169 mutations from the respective p156, p166 and/or p169 double of single mutant plasmid, (2) the 550bp Hae II/ Bam HI fragment containing the relevant 217 mutant from the respective p217 plasmid, and (3) the 3.9kb Pvu II/ Bam HI fragment containing the F50 mutation and vector sequences. The multiple mutant F50/S156/A169/L217, as well as B . amyloliquefaciens subtilisin, B . lichenformis subtilisin and the single mutant L217 were analyzed with the above synthetic polypeptides where the P-1 amino acid in the substrate was Lys, His, Ala, Gln, Tyr, Phe, Met and Leu. These results are shown in Figures 26 and 27. These results show that the F50/S156/A169/L217 mutant has substrate specificity similar to that of the B . licheniformis enzyme and differs dramatically from the wild type enzyme. Although only data for the L217 mutant are shown, none of the single mutants (e.g., F50, S156 or A169) showed this effect. Although B . licheniformis differs in 88 residue positions from B . amyloliquefaciens , the combination of only these four mutations accounts for most of the differences in substrate specificity between the two enzymes. EXAMPLE 14 Subtilisin Mutants Having Altered Alkaline Stability A random mutagenesis technique was used to generate single and multiple mutations within the B . amyloliquefaciens subtilisin gene. Such mutants were screened for altered alkaline stability. Clones having increased (positive) alkaline stability and decreased (negative) alkaline stability were isolated and sequenced to identify the mutations within the subtilisin gene. Among the positive clones, the mutants V107 and R213 were identified. These single mutants were subsequently combined to produce the mutant V107/R213. One of the negative clones (V50) from the random mutagenesis experiments resulted in a marked decrease in alkaline stability. Another mutant (P50) was analyzed for alkaline stability to determine the effect of a different substitution at position 50. The F50 mutant was found to have a greater alkaline stability than wild type subtilisin and when combined with the double mutant V107/R213 resulted in a mutant having an alkaline stability which reflected the aggregate of the alkaline stabilities for each of the individual mutants. The single mutant R204 and double mutant C204/R213 were identified by alkaline screening after random cassette mutagenesis over the region from position 197 to 228. The C204/R213 mutant was thereafter modified to produce mutants containing the individual mutations C204 and R213 to determine the contribution of each of the individual mutations. Cassette mutagenesis using pooled oligonucleotides to substitute all amino acids at position 204, was utilized to determine which substitution at position 204 would maximize the increase in alkaline stability. The mutation from Lys213 to Arg was maintained constant for each of these substitutions at position 204. A. Construction of pB0180, an E. coli-B. subtilis Shuttle Plasmid The 2.9 kb EcoRI-BamHI fragment from pBR327 (Covarrubias, L., et al. (1981) Gene 13 , 25-35) was ligated to the 3.7kb Eco RI- Bam HI fragment of pBD64 (Gryczan, T., et al. (1980) J. Bacteriol. , 141, 246-253) to give the recombinant plasmid pB0153. The unique Eco RI recognition sequence in pBD64 was eliminated by digestion with Eco RI followed by treatment with Klenow and deoxynucleotide triphosphates (Maniatis, T., et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual , Cold spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Blunt end ligation and transformation yielded pB0154. The unique Ava I recognition sequence in pBO154 was eliminated in a similar manner to yield pBO171. pB0171 was digested with Bam HI and Pvu II and treated with Klenow and deoxynucleotide triphosphates to create blunt ends. The 6.4 kb fragment was purified, ligated and transformed into LE392 cells (Enquest, L.W., et al. (1977) J. Mol. Biol. 111 , 97-120), to yield pB0172 which retains the unique Bam HI site. To facilitate subcloning of subtilisin mutants, a unique and silent Kpn I site starting at codon 166 was introduced into the subtilisin gene from pS4.5 (Wells, J.A., et al. (1983) Nucleic Acids Res. , 11 , 7911-7925) by site-directed mutagenesis. The Kpn I+ plasmid was digested with Eco RI and treated with Klenow and deoxynucleotide triphosphates to create a blunt end. The Klenow was inactivated by heating for 20 min at 68°C, and the DNA was digested with Bam HI. The 1.5 kb blunt Eco RI- Bam HI fragment containing the entire subtilisin was ligated with the 5.8 kb Nru I- Bam HI from pB0172 to yield pBO180. The ligation of the blunt Nru I end to the blunt Eco RI end recreated an Eco RI site. Proceeding clockwise around pB0180 from the Eco RI site at the 5' end of the subtilisin gene is the unique Bam HI site at the 3' end of the subtilisin gene, the chloramphenicol and neomycin resistance genes and UB110 gram positive replication origin derived from pBD64, the ampicillin resistance gene and gram negative replication origin derived from pBR327. B. Construction of Random Mutagenesis Library The 1.5 kb Eco RI- Bam HI fragment containing the B. amyloliquefaciens subtilisin gene (Wells et al., 1983) from pB0180 was cloned into M13mp11 to give M13mp11 SUBT essentially as previously described (Wells, J.A., et al. (1986) J. Biol. Chem. , 261 ,6564-6570). Deoxyuridine containing template DNA was prepared according to Kunkel (Kunkel, T.A. (1985) Proc. Natl. Acad. Sci. USA , 82 488-492). Uridine containing template DNA (Kunkel, 1985) was purified by CsCl density gradients (Maniatis, T. et al. (eds.) (1982) in Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). A primer (AvaI⁻) having the sequence ending at codon -11, was used to alter the unique Ava I recognition sequence within the subtilisin gene. (The asterisk denotes the mismatches from the wild-type sequence and underlined is the altered Ava I site.) The 5' phosphorylated Ava I primer (∼320 pmol) and ∼40 pmol (∼120µg) of uridine containing M13mp11 SUBT template in 1.88 ml of 53 mM NaCl, 7.4 mM MgCl2 and 7.4 mM Tris.HCl (pH 7.5) were annealed by heating to 90°C for 2 min. and cooling 15 min at 24°C (Fig. 31). Primer extension at 24°C was initiated by addition of 100µL containing 1 mM in all four deoxynucleotide triphosphates, and 20µl Klenow fragment (5 units/l). The extension reaction was stopped every 15 seconds over ten min by addition of 10µl 0.25 M EDTA (pH 8) to 50µl aliquots of the reaction mixture. Samples were pooled, phenol chlorophorm extracted and DNA was precipitated twice by addition of 2.5 vol 100% ethanol, and washed twice with 70% ethanol. The pellet was dried, and redissolved in 0.4 ml 1 mM EDTA, 10 mM Tris (pH 8). Misincorporation of α-thiodeoxynucleotides onto the 3' ends of the pool of randomly terminated template was carried out by incubating four 0.2 ml solutions each containing one-fourth of the randomly terminated template mixture (∼20µg), 0.25 mM of a given α-thiodeoxynucleotide triphosphate, 100 units AMV polymerase, 50 mM KCL, 10 mM MgCl₂, 0.4 mM dithiothreitol, and 50 mM Tris (pH 8.3) (Champoux, J.J. (1984) Genetics , 2 , 454-464). After incubation at 37°C for 90 minutes, misincorporation reactions were sealed by incubation for five minutes at 37°C with 50 mM all four deoxynucleotide triphosphates (pH 8), and 50 units AMV polymerase. Reactions were stopped by addition of 25 mM EDTA (final), and heated at 68°C for ten min to inactivate AMV polymerase. After ethanol precipitation and resuspension, synthesis of closed circular heteroduplexes was carried out for two days at 14°C under the same conditions used for the timed extension reactions above, except the reactions also contained 1000 units T4 DNA ligase, 0.5 mM ATP and 1 mM β-mercaptoethanol. Simultaneous restriction of each heteroduplex pool with Kpn I, Bam HI, and Eco RI confirmed that the extension reactions were nearly quantitative. Heteroduplex DNA in each reaction mixture was methylated by incubation with 80µM S-adenosylmethionine and 150 units dam methylase for 1 hour at 37°C. Methylation reactions were stopped by heating at 68°C for 15 min. One-half of each of the four methylated heteroduplex reactions were transformed into 2.5 ml competent E . coli JM101 (Messing, J. (1979) Recombinant DNA Tech. Bull. , 2 , 43-48). The number of independent transformants from each of the four transformations ranged from 0.4-2.0 x 10⁵. After growing out phage pools, RF DNA from each of the four transformations was isolated and purified by centrifugation through CsCl density gradients. Approximately 2µg of RF DNA from each of the four pools was digested with Eco RI, Bam HI and Ava I. The 1.5 kb Eco RI- Bam HI fragment (i.e., Ava I resistant) was purified on low gel temperature agarose and ligated into the 5.5 kb Eco RI- Bam HI vector fragment of pB0180. The total number of independent transformants from each α-thiodeoxynucleotide misincorporation plasmid library ranged from 1.2-2.4 x 10⁴. The pool of plasmids from each of the four transformations was grown out in 200 ml LB media containing 12.5µg/ml cmp and plasmid DNA was purified by centrifugation through CsCl density gradients. C. Expression and Screening of Subtilisin Point Mutants Plasmid DNA from each of the four misincorporation pools was transformed (Anagnostopoulos, C., et al. (1967), J. Bacteriol. , 81 , 741-746) into BG2036. For each transformation, 5µg of DNA produced approximately 2.5 x 10⁵ independent BG2036 transformants, and liquid culture aliquots from the four libraries were stored in 10% glycerol at 70°C. Thawed aliquots of frozen cultures were plated on LB/5µg/ml cmp/1.6% skim milk plates (Wells, J.A., et al. (1983) Nucleic Acids Res. , 11 , 7911-7925), and fresh colonies were arrayed onto 96-well microtiter plates containing 150 l per well LB media plus 12.5µg/ml cmp. After 1 h at room temperature, a replica was stamped (using a matched 96 prong stamp) onto a 132 mm BA 85 nitrocellulose filter (Schleicher and Scheull) which was layered on a 140 mm diameter LB/cmp/skim milk plate. Cells were grown about 16 h at 30°C until halos of proteolysis were roughly 5-7 mm in diameter and filters were transferred directly to a freshly prepared agar plate at 37°C containing only 1.6% skim milk and 50 mM sodium phosphate pH 11.5. Filters were incubated on plates for 3-6 h at 37°C to produce halos of about 5 mm for wild-type subtilisin and were discarded. The plates were stained for 10 min at 24°C with Coomassie blue solution (0.25% Coomassie blue (R-250) 25% ethanol) and destained with 25% ethanol, 10% acetic acid for 20 min. Zones of proteolysis appeared as blue halos on a white background on the underside of the plate and were compared to the original growth plate that was similarly stained and destained as a control. Clones were considered positive that produced proportionately larger zones of proteolysis on the high pH plates relative to the original growth plate. Negative clones gave smaller halos under alkaline conditions. Positive and negative clones were restreaked to colony purify and screened again in triplicate to confirm alkaline pH results. D. Identification and Analysis of Mutant Subtilisins Plasmid DNA from 5 ml overnight cultures of more alkaline active B.subtilis clones was prepared according to Birnboim and Doly (Birnboim, H.C., et al. (1979) Nucleic Acid Res. 7 , 1513) except that incubation with 2 mg/ml lysozyme proceeded for 5 min at 37°C to ensure cell lysis and an additional phenol/CHCl₃ extraction was employed to remove contaminants. The 1.5 kb Eco RI- Bam HI fragment containing the subtilisin gene was ligated into M13mp11 and template DNA was prepared for DNA sequencing (Messing, J., et al. (1982) Gene , 19 269-276). Three DNA sequencing primers ending at codon 26, +95, and +155 were synthesized to match the subtilisin coding sequence. For preliminary sequence identification a single track of DNA sequence, corresponding to the dNTPaS misincorporation library from which the mutant came, was applied over the entire mature protein coding sequence (i.e., a single dideoxyguanosine sequence track was applied to identify a mutant from the dGTPas library). A complete four track of DNA sequence was performed 200 bp over the site of mutagenesis to confirm and identify the mutant sequence (Sanger, F., et al., (1980) J. Mol. Biol. , 143 , 161-178). Confirmed positive and negative bacilli clones were cultured in LB media containing 12.5µg/mL cmp and purified from culture supernatants as previously described (Estell, D.A., et al. (1985) J. Biol. Chem. , 260 , 6518-6521). Enzymes were greater than 98% pure as analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, U.K. (1970), Nature , 227 , 680-685), and protein concentrations were calculated from the absorbance at 280 nm, (Maturbara, H., et al. (1965), J. Biol. Chem , 240, 1125-1130). Enzyme activity was measured with 200µg/mL succinyl-L-AlaL-AlaL-ProL-Phep-nitroanilide (Sigma) in 0.1M Tris pH 8.6 or 0.1 M CAPS pH 10.8 at 25°C. Specific activity (µ moles product/min-mg) was calculated from the change in absorbance at 410 nm from production of p-nitroaniline with time per mg of enzyme (E410 = 8,480 M-lcm-l; Del Mar, E.G., et al. (1979), Anal. Biochem. , 99 , 316-320). Alkaline autolytic stability studies were performed on purified enzymes (200µg/mL) in 0.1 M potassium phosphate (pH 12.0) at 37°C. At various times aliquots were assayed for residual enzyme activity (Wells, J.A., et al. (1986) J. Biol. Chem. , 261 , 6564-6570). E. Results 1. Optimization and analysis of mutagenesis frequency A set of primer-template molecules that were randomly 3'-terminated over the subtilisin gene (Fig. 31) was produced by variable extension from a fixed 5'-primer (The primer mutated a unique AvaI site at codon 11 in the subtilisin gene). This was achieved by stopping polymerase reactions with EDTA after various times of extension. The extent and distribution of duplex formation over the 1 kb subtilisin gene fragment was assessed by multiple restriction digestion (not shown). For example, production of new HinfI fragments identified when polymerase extension had proceeded past Ile110, Leu233, and Asp259 in the subtilisin gene. Misincorporation of each dNTPαs at randomly terminated 3' ends by AMV reverse transcriptase (Zakour, R.A., et al. (1982), Nature , 295 , 708-710; Zakour, R.A., et al. (1984), Nucleic Acids Res. , 12 , 6615-6628) used conditions previously described (Champoux, J.J., (1984), Genetics , 2 , 454-464). The efficiency of each misincorporation reaction was estimated to be greater than 80% by the addition of each dNTPαs to the AvaI restriction primer, and analysis by polyacrylamide gel electrophoresis. Misincorporations were sealed by polymerization with all four dNTP's and closed circular DNA was produced by reaction with DNA ligase. Several manipulations were employed to maximize the yield of the mutant sequences in the heteroduplex. These included the use of a deoxyuridine containing template (Kunkel, T.A. (1985), Proc. Natl. Acad. Sci. USA , 82 488-492; Pukkila, P.J. et al. (1983), Genetics , 104 , 571-582), in vitro methylation of the mutagenic strand (Kramer, W. et al. (1982) Nucleic Acids Res. , 10 6475-6485), and the use of Ava I restriction-selection against the wild-type template strand which contained a unique Ava I site. The separate contribution of each of these enrichment procedures to the final mutagenesis frequency was not determined, except that prior to Ava I restriction-selection roughly one-third of the segregated clones in each of the four pools still retained a wild-type Ava I site within the subtilisin gene. After Ava I restriction-selection greater than 98% of the plasmids lacked the wild-type Ava I site. The 1.5 kb Eco RI- Bam HI subtilisin gene fragment that was resistant to Ava I restriction digestion, from each of the four CsCl purified M13 RF pools was isolated on low melting agarose. The fragment was ligated in situ from the agarose with a similarly cut E . coli - B . subtilis shuttle vector, pB0180, and transformed directly into E coli LE392. Such direct ligation and transformation of DNA isolated from agarose avoided loses and allowed large numbers of recombinants to be obtained (>100,000 per µg equivalent of input M13 pool). The frequency of mutagenesis for each of the four dNTPαs misincorporation reactions was estimated from the frequency that unique restriction sites were eliminated (Table XX). The unique restriction sites chosen for this analysis, Cla I, Pvu II, and Kpn I, were distributed over the subtilisin gene starting at codons 35, 104, and 166, respectively. As a control, the mutagenesis frequency was determined at the Pst I site located in the β lactamase gene which was outside the window of mutagenesis. Because the absolute mutagenesis frequency was close to the percentage of undigested plasmid DNA, two rounds of restriction-selection were necessary to reduce the background of surviving uncut wild-type plasmid DNA below the mutant plasmid (Table XX). The background of surviving plasmid from wild-type DNA probably represents the sum total of spontaneous mutations, uncut wild-type plasmid, plus the efficiency with which linear DNA can transform E . coli . Subtracting the frequency for unmutagenized DNA (background) from the frequency for mutant DNA, and normalizing for the window of mutagenesis sampled by a given restriction analysis (4-6 bp) provides an estimate of the mutagenesis efficiency over the entire coding sequence (∼1000 bp). From this analysis, the average percentage of subtilisin genes containing mutations that result from dGTPαs, dCTPαs, or dTTPαs misincorporation was estimated to be 90, 70, and 20 percent, respectively. These high mutagenesis frequencies were generally quite variable depending upon the dNTPαs and misincorporation efficiencies at this site. Misincorporation efficiency has been reported to be both dependent on the kind of mismatch, and the context of primer (Champoux, J.J., (1984); Skinner, J.A., et al. (1986) Nucleic Acids Res. , 14 , 6945-6964). Biased misincorporation efficiency of dGTPαs and dCTPαs over dTTPαs has been previously observed (Shortle, D., et al. (1985), Genetics , 110 , 539-555). Unlike the dGTPαs, dCTPαs, and dTTPαs libraries the efficiency of mutagenesis for the dATPαs misincorporation library could not be accurately assessed because 90% of the restriction-resistant plasmids analyzed simply lacked the subtilisin gene insert. This problem probably arose from self-ligation of the vector when the dATPαs mutagenized subtilisin gene was subcloned from M13 into pB0180. Correcting for the vector background, we estimate the mutagenesis frequency around 20 percent in the dATPαs misincorporation library. In a separate experiment (not shown), the mutagenesis efficiencies for dGTPαs and dTTPαs misincorporation were estimated to be around 50 and 30 percent, respectively, based on the frequency of reversion of an inactivating mutation at codon 169. The location and identity of each mutation was determined by a single track of DNA sequencing corresponding to the misincorporated αthiodeoxynucleotide over the entire gene followed by a complete four track of DNA sequencing focused over the site of mutation. Of 14 mutants identified, the distribution was similar to that reported by Shortle and Lin (1985) except we did not observe nucleotide insertion or deletion mutations. The proportion of AG mutations was highest in the G misincorporation library, and some unexpected point mutations appeared in the dTTPαs and dCTPαs libraries. 2. Screening and Identification of Alkaline Stability Mutants of Subtilisin It is possible to screen colonies producing subtilisin by halos of casein digestion (Wells, J.A. et al. (1983) Nucleic Acids Res. , 11 , 7911-7925). However, two problems were posed by screening colonies under high alkaline conditions (>pH 11). First, B . subtilis will not grow at high pH, and we have been unable to transform an alkylophilic strain of bacillus. This problem was overcome by adopting a replica plating strategy in which colonies were grown on filters at neutral pH to produce subtilisin and filters subsequently transferred to casein plates at pH 11.5 to assay subtilisin activity. However, at pH 11.5 the casein micells no longer formed a turbid background and thus prevented a clear observation of proteolysis halos. The problem was overcome by briefly staining the plate with Coomassie blue to amplify proteolysis zones and acidifying the plates to develop casein micell turbidity. By comparison of the halo size produced on the reference growth plate (pH 7) to the high pH plate (pH 11.5), it was possible to identify mutant subtilisins that had increased (positives) or decreased (negatives) stability under alkaline conditions. Roughly 1000 colonies were screened from each of the four misincorporation libraries. The percentage of colonies showing a differential loss of activity at pH 11.5 versus pH 7 represented 1.4, 1.8, 1.4, and 0.6% of the total colonies screened from the thiol dGTPαs, dATPαs, dTTPαs, and dCTPαs libraries, respectively. Several of these negative clones were sequenced and all were found to contain a single base change as expected from the misincorporation library from which they came. Negative mutants included A36, E170 and V50. Two positive mutants were identified as V107 and R213. The ratio of negatives to positives was roughly 50:1. 3. Stability and Activity of Subtilisin Mutants at Alkaline pH Subtilisin mutants were purified and their autolytic stabilities were measured by the time course of inactivation at pH 12.0 (Figs. 32 and 33). Positive mutants identified from the screen (i.e., V107 and R213) were more resistant to alkaline induced autolytic inactivation compared to wild-type; negative mutants (i.e., E170 and V50) were less resistant. We had advantageously produced another mutant at position 50 (F50) by site-directed mutagenesis. This mutant was more stable than wild-type enzyme to alkaline autolytic inactivation (Fig. 33) At the termination of the autolysis study, SDS-PAGE analysis confirmed that each subtilisin variant had autolyzed to an extent consistent with the remaining enzyme activity. The stabilizing effects of V107, R213, and F50 are cumulative. See Table XXI. The double mutant, V107/R213 (made by subcloning the 920 bp Eco RI- Kpn I fragment of pB0180V107 into the 6.6 kb Eco RI- Kpn I fragment of pB0180R213), is more stable than either single mutant. The triple mutant, F50/V107/R213 (made by subcloning the 735 bp Eco RI- Pvu II fragment of pF50 (Example 2) into the 6.8 kb Eco RI- Pvu II fragment of pB0180/V107, is more stable than the double mutant V107/R213 or F50. The inactivation curves show a biphasic character that becomes more pronounced the more stable the mutant analyzed. This may result from some destablizing chemical modification(s) (eg., deamidation) during the autolysis study and/or reduced stabilization caused by complete digestion of larger autolysis peptides. These alkaline autolysis studies have been repeated on separately purified enzyme batches with essentially the same results. Rates of autolysis should depend both on the conformational stability as well as the specific activity of the subtilisin variant (Wells, J.A., et al. (1986), J. Biol. Chem. , 261 , 6564-6570). It was therefore possible that the decreases in autolytic inactivation rates may result from decreases in specific activity of the more stable mutant under alkaline conditions. In general the opposite appears to be the case. The more stable mutants, if anything, have a relatively higher specific activity than wild-type under alkaline conditions and the less stable mutants have a relatively lower specific activity. These subtle effects on specific activity for V107/R213 and F50/V107/R213 are cumulative at both pH 8.6 and 10.8. The changes in specific activity may reflect slight differences in substrate specificity, however, it is noteworthy that only positions 170 and 107 are within 6A of a bound model substrate (Robertus, J.D., et al. (1972), Biochemistry 11 , 2438-2449). Relationship between relative specific acitivity at pH 8.6 or 10.8 and alkaline autolytic stability Enzyme Relative specific activity Alkaline autolysis half-time (min) pH 8.6 pH 10.8 Wild-type 100±1 100±3 86 Q170 46±1 28±2 13 V107 126±3 99±5 102 R213 97±1 102±1 115 V107/R213 116±2 106±3 130 V50 66±4 61±1 58 F50 123±3 157±7 131 F50/V107/R213 126±2 152±3 168 F. Random Cassette Mutagenesis of Residues 197 through 228 Plasmid pΔ222 (Wells, et al. (1985) Gene 34 , 315-323) was digested with Pst I and Bam HI and the 0.4 kb Pst I/ Bam HI fragment (fragment 1, see Fig. 34) purified from a polyacrylamide gel by electroelution. The 1.5 kb Eco RI/ Bam HI fragment from pS4.5 was cloned into M13mp9. Site directed mutagenesis was performed to create the A197 mutant and simultaneously insert a silent Sst I site over codons 195-196. The mutant Eco RI/ Bam HI fragment was cloned back into pBS42. The pA197 plasmid was digested with Bam HI and Sst I and the 5.3 kb Bam HI/ Sst I fragment (fragment 2) was purified from low melting agarose. Complimentary oligonucleotides were synthesized to span the region from Sst I (codons 195-196) to Pst I (codons 228-230). These oligodeoxynucleotides were designed to (1) restore codon 197 to the wild type, (2) re-create a silent Kpn I site present in pΔ222 at codons 219-220, (3) create a silent Sma I site over codons 210-211, and (4) eliminate the Pst I site over codons 228-230 (see Fig. 35). Oligodeoxynucleotides were synthesized with 2% contaminating nucleotides at each cycle of synthesis, e.g., dATP reagent was spiked with 2% dCTP, 2% dGTP, and 2% dTTP. For 97-mers, this 2% poisoning should give the following percentages of non-mutant, single mutants and double or higher mutants per strand with two or more misincorporations per complimentary strand: 14% non-mutant, 28% single mutant, and 57% with ≧2 mutations, according to the general formula where µ is the average number of mutations and n is a number class of mutations and f is the fraction of the total having that number of mutations. Complimentary oligodeoxynucleotide pools were phosphorylated and annealed (fragment 3) and then ligated at 2-fold molar excess over fragments 1 and 2 in a three-way ligation. E . coli MM294 was transformed with the ligation reaction, the transformation pool-grown up over night and the pooled plasmid DNA was isolated. This pool represented 3.4 x 10⁴ independent transformants. This plasmid pool was digested with Pst I and then used to retransform E . coli . A second plasmid pool was prepared and used to transform B . subtilis (BG2036). Approximately 40% of the BG2036 transformants actively expressed subtilisin as judged by halo-clearing on casein plates. Several of the non-expressing transformants were sequenced and found to have insertions or deletions in the synthetic cassettes. Expressing BG2036 mutants were arrayed in microtiter dishes with 150µl of LB/12.5µg/mL chloramphenicol (cmp) per well, incubated at 37°C for 3-4 hours and then stamped in duplicate onto nitrocellulose filters laid on LB 1.5% skim milk/5µg/mL cmp plates and incubated overnight at 33°C (until halos were approximately 4-8 mm in diameter). Filters were then lifted to stacks of filter paper saturated with 1 x Tide commercial grade detergent, 50 mM Na₂CO₃, pH 11.5 and incubated at 65°C for 90 min. Overnight growth plates were Commassie stained and destained to establish basal levels of expression. After this treatment, filters were returned to pH7/skim milk/20µg/mL tetracycline plates and incubated at 37°C for 4 hours to overnight. Mutants identified by the high pH stability screen to be more alkaline stable were purified and analyzed for autolytic stability at high pH or high temperature. The double mutant C204/R213 was more stable than wild type at either high pH or high temperature (Table XXII). This mutant was dissected into single mutant parents (C204 and R213) by cutting at the unique Sma I restriction site (Fig. 35) and either ligating wild type sequence 3' to the Sma I site to create the single C204 mutant or ligating wild type sequence 5' to the SmaI site to create the single R213 mutant. Of the two single parents, C204 was nearly as alkaline stable as the parent double mutant (C04/R213) and slightly more thermally stable. See Table XXII. The R213 mutant was only slightly more stable than wild type under both conditions (not shown). Another mutant identified from the screen of the 197 to 228 random cassette mutagenesis was R204. This mutant was more stable than wild type at both high pH and high temperature but less stable than C204. G. Random Mutagenesis at Codon 204 Based on the above results, codon 204 was targeted for random mutagenesis. Mutagenic DNA cassettes (for codon at 204) all contained a fixed R213 mutation which was found to slightly augment the stability of the C204 mutant. Plasmid DNA encoding the subtilisin mutant C204/R213 was digested with Sst I and Eco RI and a 1.0 kb Eco RI/ Sst I fragment was isolated by electro-elution from polyacrylamide gel (fragment 1, see Fig. 35). C204/R213 was also digested with Sma I and Eco RI and the large 4.7 kb fragment, including vector sequences and the 3' portion of coding region, was isolated from low melting agarose (fragment 2, see Fig. 36). Fragments 1 and 2 were combined in four separate three-way ligations with heterophosphorylated fragments 3 (see Figs. 36 and 37). This heterophosphorylation of synthetic duplexes should preferentially drive the phosphorylated strand into the plasmid ligation product. Four plasmid pools, corresponding to the four ligations, were restricted with Sma I in order to linearize any single cut C204/R213 present from fragment 2 isolation, thus reducing the background of C204/R213. E . coli was then re-transformed with Sma I-restricted plasmid pools to yield a second set of plasmid pools which are essentially free of C204/R213 and any non-segregated heterduplex material. These second enriched plasmid pools were then used to transform B . subtilis (BG2036) and the resulting four mutant pools were screened for clones expressing subtilisin resistant to high pH/temperature inactivation. Mutants found positive by such a screen were further characterized and identified by sequencing. The mutant L204/R213 was found to be slightly more stable than the wild type subtilisin. See Table XXII. Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.

Claims: A subtilisin mutant derived by the substitution of at least one amino acid residue of a precursor subtilisin with a different amino acid, so that the subtilisin mutant has at least one property which is different from the same property of the precursor subtilisin, characterised by the substitution at one or more of Tyr21, Thr22, Ser24, Asp36, Ala45, Gly46, Ala48, Ser49, Met50, Asn77, Ser87, Lys94, Val95, Leu96, Ile107, Gly110, Met124, Lys170, Tyr171, Pro172, Asp197, Met199, Ser204, Lys213, His67, Leu135, Gly97, Ser101, Gly102, Glu103, Gly127, Gly128, Pro129, Tyr214, and Gly215 of Bacillus amyloliquefaciens subtilisin and equivalent amino acid residues in other precursor subtilisins. A subtilisin mutant having an amino acid sequence derived from the amino acid sequence of a precursor subtilisin by the substitution of more than one amino acid residue of said amino acid sequence of said precursor subtilisin by a different amino acid, so that the subtilisin mutant has at least one property which is different from the same property of the precursor subtilisin, characterized by substitutions at more than one of Tyr21, Thr22, Ser24, Asp32, Ser33, Asp36, Ala45, Ala48, Ser49, Met50, Ser87, Lys94, Val95, Tyr104, Ile107, Gly110, Met124, Ala152, Asn155, Glu156, Gly166, Gly169, Lys170, Tyr171, Pro172, Phe189, Asp197, Met199, Ser204, Lys213, Tyr217, Ser221, Met222, His67, Leu135, Gly97, Ser101, Gly102, Glu103, Gly127, Gly128, Pro129, Tyr214, and Gly215 of Bacillus amyloliquefaciens subtilisin and equivalent amino acid residues in other precursor subtilisins, with the proviso that when substitution is made at any residue in the group Asp32, Ser33, Tyr104, Ala152, Asn155, Glu156 Gly166, Gly169, Phe189, Tyr217 and Met222 a substitution is also made at at least one specified position not of that group. The mutant of claim 2 wherein said combinations are selected from Thr22/Ser87, Ser24/Ser87, Ala45/Ala48, Ser49/Lys94, Ser49/Val95, Met50/Val95, Met50/Gly110, Met50/Met124, Met50/Met222, Met124/Met222, Tyr21/Thr22, Met50/Met124/Met222, Tyr21/Thr22/Ser87, Met50/Glu156/Gly166/Tyr217, Met50/Glu156/Tyr217, Ile170/Lys213, Ser204/Lys213, Met50/Ile107/Lys213 and Ser24/Met50/Ile107/Glu156/Gly166/Gly169/Ser204/Lys213/Gly215/Tyr217. A subtilisin mutant derived by the deletion of one or more amino acid residues in a precursor subtilisin equivalent to 161-164 in B. amyloliquefaciens subtilisin, said deletion being made alone or in combination with substitutions in the amino acid sequence of the precursor subtilisin, and producing at least one property which is different from the same property of the precursor subtilisin. A subtilisin mutant having altered substrate specificity when compared to a precursor subtilisin, the mutant being derived by the substitution of a different amino acid at the residue equivalent to Leu+126 of B. amyloliquefaciens subtilisin, alone or in combination with other substitutions or deletions in the amino acid sequence of the precursor subtilisin. A subtilisin mutant having altered substrate specificity when compared to a precursor subtilisin, the mutant being derived by the substitution of a different amino acid at the residue equivalent to Asp+99 in B. amyloliquefaciens subtilisin, alone or in combination with other substitutions or deletions in the amino acid sequence of the precursor subtilisin. A DNA sequence encoding the mutant of any one of the preceding claims. An expression vector containing the mutant DNA sequence of claim 7. A host cell transformed with the expression vector or claim 8.

Inventors: BOTT RICHARD RAY, CALDWELL ROBERT MARK, CUNNINGHAM BRIAN C, ESTELL DAVID AARON, POWER SCOTT DOUGLAS, WELLS JAMES ALLEN, BOTT, RICHARD RAY, CALDWELL, ROBERT MARK, CUNNINGHAM, BRIAN C., ESTELL, DAVID AARON, POWER, SCOTT DOUGLAS, WELLS, JAMES ALLEN
Applicants: GENENCOR INT, GENENCOR INTERNATIONAL, INC.
Application year: 1987
Main classification: C12N15
IPC codes: C12N9, C12N15, C12R1, C11D3, C12N1