DK221787A - Ikke-humane carbonylhydrolasemutanter, dna-sekven-ser og vektorer der koder for saadanne og vaerter der er transformeret med saadanne vektorer - Google Patents

Description

NON-HUMAN CARBONYL HYDROLASE MUTANTS,

DNA SEQUENCES AND VECTORS ENCODING SAME AND HOSTS TRANSFORMED WITH SAID VECTORS

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 (ThrSl-^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 crosslinked 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,

Glyl66 with 9 different amino acids and Glyl69 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 ø-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. antvloliauefaciens 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-l binding subsite of B. amylolicnief aciens 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. amvlolicruefaciens subtilisin are equivalent residues which (1) can be mutated in a similar manner to that described for B. amvlol icruef aciens subtilisin, or (2) can be used as a replacement amino acid residue in B. amvlol icruefaciens subtilisin. Figure 5C depicts conserved residues of B. amvlolicruefaciens 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. amvloliquefaciens subtilisin.

Figure 11 depicts the construction of mutations between codons 122 and 127 of B. amylolicmefaciens 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. amvloliauefaciens subtilisin.

Figure 14 depicts the effect of hydrophobicity of the P-1 substrate side-chain on the kinetic parameters of wild-type B. amvlolicruefaciens subtilisin.

Figure 15 depicts the effect of position 166 side-chain substitutions on P-1 substrate specificity. Figure 15Ά 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 7-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 Ilel66 and wild-type (Glyl66) B. amyloliquefaciens subtilisin against a series of alphatic and aromatic substrates. Each bar represents 5 the difference in log kcat/Km for Ilel66 minus wild-type (Glyl66) subtilisin.

Figure 18 depicts the construction of mutations at codon 169 of B. amyloliquefaciens subtilisin. 10

Figure 19 depicts the construction of mutations at codon 104 of 13. amyloliquefaciens subtilisin.

Figure 20 depicts the construction of mutations at 15 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. 20

Figure 22 depicts the construction of mutations at codon 217 for B. amyloliquefaciens subtilisin.

Figure 23 depicts the kcat/Km versus pH profile for 25 mutations at codon 156 and 166 in B. amyloliquefaciens subtilisin. v

Figure 24 depicts the kcat/Km versus pH profile for mutations at codon 222 in B. amyloliquefaciens 3Q subtilisin. 35

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 misin-corporation of "-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. amvlolicmefaciens 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 C-X 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 a-aminoacylpeptide hydrolase, peptidylamino-acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxy-peptidase, 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-histidine-serine. In the chymotrypsin related proteases tne 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. amvlolicruefaciens subtilisin are identified for substitution, insertion or deletion. These amino acid position numbers refer to those assigned to the B. amvloliauefaciens 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. amvlolicruefaciens subtilisin. A residue (amino acid) of a precursor carbonyl hydrolase is equivalent to a residue of B. amvlolicruefaciens 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. amvlolicruefaciens 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. amvlol icruefaciens 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. amvloliauefaciens 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. amvloliauefaciens B. subtilisin var. 1168 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. amvloliauefaciens 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. amvloliauefaciens subtilisin. Thus, in thermitase the equivalent amino acid of Tyr217 in B. amvloliauefaciens subtilisin is the particular lysine shown beneath Tyr217.

In Fig. 5A, the equivalent amino acid at position 217 in B. amvloliquefaciens 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. amvloliouefaciens 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. amvlolioue-faciens 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. amvloliouefaciens subtilisin (N on N, CA on CA, C on C, and 0 on 0) are within 0.13nm and preferably O.lnm 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. amvlolicruefaciens 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. amvlol icruef aciens 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. amvlol icruef aciens 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. amvloliouefaciens 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 - 19-

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 1168 (EPO Publication No. 0130756).

Host cells are transformed or transfected with vectors constructed using recombinant DNA technigues. 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 preseguence 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. 01^0756) 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. amvlolicruefaciens subtilisin shown in Table I. The wild type amino acid sequence and DNA sequence of B. amyloliquefaciens subtilisin is shown in Fig. 1.

TABLE I

Residue _Replacement Anting Acid_

Tyr21 F ft

Thr22 C

Ser24 C

Asp32 Q S

Ser3 3 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

Tyrl04 ACDEFGHIKLMNPQRSTVW

Ilel07 V

GlyllO C R

Metl24 I L

Asnl55 A D H Q T

Glul56 Q S

Gly166 CEILMPSTWY

Gly169 CDEFHIKLMNPQRTVWY

Lysl70 E R

Tyrl71 F

Prol72 E Q

Phel89 ACDEGHIKLMNPQRSTVWY

Aspl9 7 R A

Metl99 1

Ser204 C R L P

Lys213 R T

Tyr217 ACDEFGHIKLMNPQRSTVW

Ser221 A C

The different amino acids substituted are represented in Table I by the following single letter designations:

Amino acid or residue 3-letter 1-letter thereof symbol symbol

Alanine Ala A

Glutamate Glu E

Glutamine Gin Q

Aspartate Asp D

Asparagine Asn N

Leucine Leu L

Glycine Gly G

Lysine Lys κ

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. amvloliouefaciens 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.

TABLE II

Residue Replacement Amino Acid(si

Tyr-21 L

Thr22 K

Ser24 A

Asp32

Ser33 G

Gly46

Ala48

Ser49

Met50 L K I V

Asn77 D

Ser87 N

Lys9 4 R Q

Val95 L I

Tyrl04

Metl24 K A

Alal52 C L I T M

Asnl55

Glul56 A T M L Y

Glyl66

Glyl69

Tyrl71 K R E Q

Prol72 D N

Phel89

Tyr217

Ser221

Met222

Each of the mutant subtilisins in Table I contain the replacement of a single residue of the B. amvlolicmefaciens amino acid sequence. These particular residues were chosen to probe the influence of such substitutions on various properties of B. amvlolicruefacien subtilisin.

Thus, the inventors have identified Metl24 and Met222 as important residues which if substituted with another amino acid produce a mutant subtilisin with enhanced oxidative stability. For Metl24, Leu and lie 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 Tyrl04, Alal52, Glul56, Glyl66, Glyl69, Phel89 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. amylolicmefaciens 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.

Atomic Coordinates for the Apoenzyme Form of JB, Amvloliquefaciens Subtilisin to 1.8AResolution

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) Carlsbera 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 Glyl66, Glyl69 and Alal52. These amino acids were identified as residues within the S-l subsite. As seen in Fig. 3, which is a stereoview of the S-l subsite, Glyl66 and Glyl69 occupy positions at the bottom of the S-l subsite, whereas Alal52 occupies a position near the top of S-l, close to the catalytic Ser221.

All 19 amino acid substitutions of Glyl66 and Glyl69 have been made. As will be indicated in the examples which follow, the preferred replacement amino acids for Glyl66 and/or Glyl69 will depend on the specific amino acid occupying the P-1 position of a given substrate.

The only substitutions of Alal52 presently made and analyzed comprise the replacement of Alal52 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-l substrate amino acid, Tyrl04 has been identified as being involved with P-4 specificity. Substitutions at Phel89 and Tyr217, however, are expected to respectively effect P-21 and P-l' specificity.

The catalytic activity of subtilisin has also been modified by single amino acid substitutions at Asnl55. The catalytic triad of subtilisin is shown in Fig. 4. As can be seen, Ser221, His64 and Asp32 are 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.

Asnl55 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 Asnl55 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 amvlolicfuefaciens subtilisin residues Asp36, Ilel07, Lysl70, 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 Lysl70 with Glu each resulted in a mutant enzyme having a lower alkaline stability as compared to the wild type subtilisin. When Ilel07 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, Glul56, Glyl66, Glyl69 and Tyr217. Specifically the following particular substitutions result in an increased alkaline stability: Ser24C, MetSOF, Glyl56Q or S, Glyl66A, H, K, N or Q, Glyl69S or A, and Tyr217F, K, R or L. The mutant MetSOV, 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 Aspl97 and Met222. Particular mutants include Aspl97(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 Metl99 and Tyr21. These latter two residues are also believed to be important for alkaline stability. Mutants at these residues include 1199 and F21.

The amino acid sequence of B. amvlolicruefaciens 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. amvlolicruefaciens 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 g. amvlolicruefaciens 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. amvlol icruef aciens 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. amvlolicruifaciens 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 (lie 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. amvlolicruifaciens 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, Ilel07, Glul56, Glyl66, Glyl69, Ser204, Lys213, Gly215, and Tyr217.

TABLE IV

Triple, Quadruple

Double Mutants _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/A161- S156/K166 164/I165/S166/A169/R170] S156/N166 L204/R213 S156/A169 R213/204A, E, Q, D, N, G, K,

A166/A222 V, R, T, P, I, H, F, Y, W

A166/C222 or H 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, Ilel07, Leul26 and Leul35. Mutation of His67 should alter the S-1' subsite, thereby altering the specificity of the mutant for the P-l' 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.

Ilel07 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. Ilel07 was also identified by molecular modeling from product inhibitor complexes.

The S-2 binding site includes the Leul26 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-l, S-l', S-2', S-31 and cleavage would be forced to occur after the amino terminal peptide.

Leul35 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 these 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. amvloliouifaciens 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, SerlOl interacts with Asp99 in B. amvliouefaciens subtilisin to stabilize the same main chain turn. Alterations at this residue should alter the 101-103 main chain direction.

Mutations at Glul03 are also expected to affect the 101-103 main chain direction.

The side chain of Glyl02 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. Gly 127 is positioned such that its side chain interacts with the S-l 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 Leul26 would be expected to produce that result.

The Prol29 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/A161-164/I165/S166/A169/ R170). This produced the following results:

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.

TABLE VI

Substitution/Insertion/Deletion

Residues

His67 Alal52

Leul26 Alal53

Leul35 Glyl54

Gly9 7 Asnl55

Asp99 Glyl56

SerlOl Glyl57

Glyl02 Glyl60

Glul03 ThrI58

Leul26 Serl59

Glyl27 Seri 61

Glyl28 Serl62

Prol29 Serl63

Tyr214 Thrl64

Gly215 Vall65

Glyl66 Glyl69

Tyrl67 Lysl70

Prol68 Tyrl71

Prol72

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, ImM 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% NaDodSO^, 5% glycerol and bromophenol blue) and disassociated at 95°C for 3 minutes.

The samples were electrophoresed on discontinuous polyacrylamide gels (Kyte, J.f et al. (1983) 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 (IBP, 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 280nra. 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/l-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 <~lnM each amino acid) were dried, hydrolyzed in vacuo with 100 ul 6N HC1 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.

TABLE VII

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) Met 12 4 is also oxidized leading to the loss of peptide 7 and the accumulation of peptide 6. Thus oxidation of g. amvlolicruifaciens subtilisin with the peracid, diperdocecanoic acid leads to the specific oxidation of methionine at residues 50 and 124. EXAMPLE 2

Substitution at Met50 and Metl24 in Subtilisin Met2220_

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 Savler's Z. Phvsiol. Chem. 364 1537-1540), B. amvlosacchariticus (Markland, F.S., et al. (1967) J. Biol. Chem. 242 5198-5211) and B. subtilis (Stahl, M.L., et al. (1984) J. Bacterlol. 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 required 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 ρΔ50 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, Ml3mpll-SUBT was used for heteroduplex synthesis (Adelman, et al (1983), DNA 2, 183-193). Following transfection of JM101 (ATCC 33876), the 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was subcloned from M13mpll 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 (pa50, line 4) , the resulting plasmid pool was digested with Kpnlf 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 Kpnl site. Kpnl plasmids were sequenced and confirmed the ρΔ50 sequence. Asterisks in Figure 11 indicate the bases that are mutated from the wid type sequence (line 4). ρΔ50 (line 4) was cut with StuI and EcoRI and the 0.5 Kb fragment containing the 5' half of the subtilisin gene was purified (fragment 1). ΡΔ50 (line 4) was digested with Kpnl and EcoRI 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 2 A was followed in substantial detail except that the mutagenesis primer of Figure 11, line 7 was used and restriction-purification for the EcoRV site in ΡΔ124 was used. In addition, the DNA cassette (shaded sequence, Figure 11, line 6) contained the triplet ATT for codon 124 which encodes lie and CTT for Leu. Those plasmids which contained the substitution of lie for Metl24were designeated pii24. The mutant subtilisin was designated 1124. C. Construction of Various F50/I124/0222 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 Avail to PvuII fragment from pF50; the 1124 mutation was contained on a 260 bp PvuII to Avail fragment from pi 124; and the Q222 mutation was contained on 2.7 kb Avail to Avail 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 Avail site at position 798 in the wild-type subtilisin gene was eliminated by the 1124 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 0222 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 Metl24 to 1124 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-l 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. Amvloliquefaciens

Wild-type subtilisin was purified from B. subtilis culture supernatants expressing the B. amvlolicrue-faciens 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 PI 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).

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 Enzymoloqy (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, aG^. A plot of the log kcat/Km versus the hydrophobicity of the PI 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., Enzvme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287; Harper, J.W., et al. (1984) Biochemistry. 23. 2995-3002), suggests that the PI 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 li. 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 PI 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-l binding subsite. To test this hypothesis, hydrophobic amino acid substitutions of Glyl66 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-l 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 PI 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 SacI and Xmal 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 ΡΔ166 (Figure 13, line 2). pal66 was cut open with SacI and Xmal. 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 ρΔΐ66 (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. amvloliouefaciens 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-l subsite, position 166 mutants were kinetically analyzed versus PI 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., aaG^), 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 Glyl66 (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 £-hydroxyl group, β- or 7-aliphatic branching, cause large decreases in kcat/Km for larger PI 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 0-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 1166 causes a lowering of kcat/Km between two and six fold toward Met, Phe and Tyr substrates. Inserting a 7-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 7-branched appears to induce less steric hindrance toward the Phe P-1 substrate than 0-branching, as evidenced by the 100 fold decrease in kcat/Km for the Phe substrate in going from L166 to 1166.

Reductions in kcat/Km resulting from increases in side chain size in the S-l subsite, or specific structural features such as β- and 7-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. Biochera. 53. 537-572). Catalytic efficiency for the Ala substrate reaches a maximum for 1166, 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 /9-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 1166]. The Tyr substrate is most efficiently utilized by wild type enzyme (Glyl66), and there is a steady decrease as one proceeds to large position 166 side-chains. The ^-branched and 7-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 PI substrate [i.e., 1166/Ala substrate, L166/Met substrate, A166/Phe substrate, Glyl66/Tyr substrate]. The combined volumes for these optimal pairs may approximate the volume for productive binding in the S-l subsite. For the optimal pairs, Glyl66/Tyr substrate, A166/Phe substrate, L166/Met substrate, V166/Met substrate, and 1166/Ala substrate, the combined volumes are 266,295,313,339 and 261 A , respectively. Subtracting the volume of the peptide backbone from each pair (i.e., two times the volume of 3 glycine), an average side-chain volume of 160±32A 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 100A of excess 3 volume. (100A 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 Glyl66 to 1166 for the Ala substrate (net of ten-fold), from Glyl66 to L166 for the Met substrate (net of ten-fold) and from Glyl66 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 (l/r6) and because of the weak nature of these attractive forces (Jencks, W.P., Catalysis in Chemistry and Enzvmolocrv (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-l 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 Serl66 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 3 (99 versus 118A ). 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 1166 mutation illustrates particularly well that large changes in specificity can be produced by altering the structure and hydrophobicity of the S-l 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 1166 enzyme becomes poorer against larger aromatic substrates of increasing size (e.g., 1166 is over 1,000 fold worse against the Tyr substrate than is Glyl66). We interpret the increase in catalytic efficiency toward the small hydrophobic substrates for 1166 compared to Glyl66 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 1166 versus Glyl66 is attributed to steric repulsion.

The specificity differences between Glyl66 and 1166 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 1166 versus Glyl66 in subtilisin. EXAMPLE 4

Substitution of Ionic Amino Acids for Glvl66_

The construction of subtilisin mutants containing the substitution of the ionic amino acids Asp, Asn, Gin, 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. ΡΔ166, described in Example 3, was digested with SacI and Xmal. 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 Glyl66. 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.

TABLE IX P-1 Substrate (kcat/Km x 10-4)

Position 166 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

Gin (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 Glyl66 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 Glveine at Position 169 The substitution of Glyl69 in B. amvlolicruefaciens subtil isin 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 Glyl69 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.

TABLE X

Effect of Serine and Alanine Mutations at Position 169 on P-1 Substrate Specificity P-1 Substrate fkcat/Km x 10~4Ϊ Position 169 Phe Leu Ala Arq

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 Glyl69 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

Tyrl04 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 Hindlll site and a frame shift mutation at codon 104. Restriction-purification for the unique Hindlll site facilitated the isolation of the mutant sequence (line 4) . Restriction-selection against this Hindlll 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 I

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.

TABLE XI

kcat _Km_ _Kcat/Km_

Substrate WT H104 WT H104 WT H104 sAAPFpNA 50.0 22.0 1.4xl0~ 4 7.1xl0~4 3.6xl0 5 3.1xl04 sAAPApNA 3.2 2.0 2.3xl0~ 4 1.9χ1θ"3 1.4xl0 4 lxlO3 sFAPFpNA 26.0 38.0 1.8xl0~ 4 4.1xl0-4 1.5xl0 5 9.1xl04 sFAPApNA 0.32 2.4 7.3xl0~ 5 1.5xl0-4 4.4xl0 3 1.6xl04

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 Alal52

Alal52 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 Kpnl site. Other mutant primers (shaded sequences Figure 20; S152, line 5 and G152, line 6) mutated the new Kpnl site away and such mutants were isolated using the restriction-selection procedure as described above for loss of the Kpnl 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.

TABLE XII P-1 Substrate (kcat/KmxlO-4)

Position 152 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 Alal52 with Ser or Gly causes a dramatic reduction in catalytic efficiencies across all substrates tested. This suggests Alal52, at the top of the S-l subsite, may be the optimal amino acid because Ser and Gly are homologous Ala substitutes. EXAMPLE 8

Substitution at Position 156

Mutants containing the substitution of Ser and Gin for Glul56 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 Glyl66, single mutations at Glul56 were obtained.

The plasmid pa 166 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 pl66 in Figure 21 thus represents the mutant plasmids of Examples 3 and 4. In this particular example, pl66 contains the wild type Glyl66.

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 SacI-BamHI 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 Konl 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-CCOGGT-AAC-GAA-3 ' to produce the mutation. Enrichment for the mutant sequence was accomplished by restriction with Kpnl. purification and self ligation. The mutant sequence containing the Kpnl site was confirmed by direct plasmid sequencing to give pV152. pV152 (-1 μg) was digested with Kpnl and treated with 2 units of DNA polymerase I large fragment (Klenow fragment from Boeringer-Mannheim) plus 50 μΚ deoxynucleotide triphosphates at 37eC for 30 min. This created a blunt end that terminated with codon 151. The DNA was extracted with 1:1 volumes phenol and CHC13 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, Gin, 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

Glyl66 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 Glyl66.

The 156 substituents were Gin and Ser. The Gin and Ser substitutions at Glyl56 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 SacI-BamHI fragment from the relevant pl56 plasmid containing the 0.6kb SacI-BamHI fragment from the relevant pl66 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.

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, Gin, Met and Lys as the p-l substrate amino acid. This permitted comparisons between side-chains that were more sterically similar but differed in charge (e.g., Glu versus Gin, Lys versus Met). Similarly, mutant enzymes were assayed against homologous P-l substrates that were most sterically similar but differed in charge (Table XIV).

TABLE XIV

Kinetics of Position 156/166 Subtilisins Determined for Different PI Substrates

Maximum difference: log kcat/Km (log 1/Km)(d) 3.5 (3.0) 1.8 (1.4) 2.3 (2.2) -1.3 (-1.0)

Footnotes to Table XIV; ^ B. subtilis. BG 2036, expressing indicated variant subtilisin were fermented and enzymes purified as previously described (Estell, et al. (1985) J. Biol. Chem. 260. 6518-6521). Wild type subtilisin is indicated (wt) containing Glul56 and Glyl66. ^ Net charge in the P-1 binding site is defined as the sum of charges from positions 156 and 166 at pH 8.6. / c} -1 v ' Values for kcat(s ) and Km(M) were measured in 0.1M Tris pH 8.6 at 25eC as previously describedJ against P-1 substrates having the form succinyl-L-AlaL-AlaL-ProL-[X]-p-nitroanilide, where X is the indicated P-1 amino acid. Values for log 1/Km are shown inside parentheses. All errors in determination of kcat/Km and 1/Km are below 5%. ^ Because values for Glul56/Aspl66(D166) are too small to determine accurately, the maximum difference taken for GluP-1 substrate is limited to a charge range of +1 to -1 charge change. n.d. = not determined

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 (AGT). Mutations at position 156 and 166 produce changes in catalytic efficiency toward Glu, Gin, 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 Glnl56/Lysl66 (Q156/K166) versus Glul56/Metl66 (Glul56/M166)] dramatically increased kcat/Km toward the Glu P-1 substrate (up to 3100 fold), and decreased the catalytic efficiency toward the Lys p-i 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 are 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, Gin (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 Gin P-l substrates (Figure 28C), and diverge for Lys and Met P-l substrates (Figure 28D). Although less pronounced effects are seen in log kcat, the effects of P-l charge on log kcat parallel those seen in log 1/Km and become larger as the P-l 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-l binding-site charge on substrate preference can be estimated from the differences in slopes between the charged and neutral isosteric P-l substrates (Figure 28B). The average change in substrate preference (Alog 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.

TABLE XV

Differential Effect on Binding Site Charge on log kcat/Kxn or (log 1/Km) . for P-1 Substrates that Differ in Charge a'

Change in P-1 Binding ilog Xcat/Km Ul0g 1/Km) __Site Charge1 ' 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 or (log 1/Km) per unit charge change 1.1 (1.0) 1.0 (0.8) 2.1 (1.5) ^ The difference in the slopes of curves were taken between the P-1 substrates over the charge interval given for log (kcat/Km) (Figure 28A, B) and (log 1/Km) (Figure 28C, D) . Values represent the differential effect a charge change has in distinguishing the substrates that are compared. ^ Charge in P-1 binding site is defined as the sum of charges from positions 156 and 166.

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.

TABLE XVI

Effect of Salt Bridge Formation Between Enzyme (a) and Substrate on PI Substrate Preference

Footnotes to Table XVI: ^ Molecular modeling shows it is possible to form a salt bridge between the indicated charged P-1 substrate and a complementary charge in the P-1 binding site of the enzyme at the indicated position changed.

Enzymes compared have sterically similar amino acid substitutions that differ in charge at the indicated position. ' ' The P-1 substrates compared are structurally similar but differ in charge. The charged P-1 substrate is complementary to the charge change at the position indicated between enzymes 1 and 2. ^ Date from Table XIV was used to compute the difference in log (kcat/Km) between the charged and the non-charged P-1 substrate (i.e., the substrate preference). The substrate preference is shown separately for enzyme 1 and 2. fa} v ' The difference in substrate preference between enzyme 1 (more highly charged) and enzyme 2 (more neutral) represents the rate change accompanying the electrostatic interaction.

The difference between catalytic efficiencies (i.e., alog kcat/Km) for the charged and neutral P-1 substrates (e.g., Lys minus Met or Glu minus Gin) give the substrate preference for each enzyme. The change in substrate preference (aalog kcat/Km) between the charged and more neutral enzyme homologs (e.g., Glul56/Glyl66 minus Glnl56(Q156)/Glyl66) 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 aalog 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 EcoRV restriction site was used for restriction-purification of ρΔ217.

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 -4 mutant has a kcat of 277 5' and a Km of 4.7x10 with a kcat/Km ratio of 6xl05. 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. amvlolicruefacien 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 Sau3A site.) The B. amvlolicruefaciens subtilisin gene on a 1.5 kb EcoRI-BAMHI fragment from pS4.5 was cloned into M13mpll and single stranded DNA was isolated. This template (M13mpllSUBT) 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 MstI 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 Sau3A 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 EcoRl-BamHI 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 Sau3A site at codon 23 that was eliminated by the mutagenesis primer. Two out of 16 plasmid preparations had lost the wild type Sau3A 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 Clal site that separated the single parent cystine codons. Specifically, the 500 bp EcoRl-Clal fragment containing the 5' portion of the subtilisin gene (including codons 22 and 24) was ligated with the 4.7 kb Clal-EcoRI 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, Sau3A minus; Cys87, Mstl plus). Plasmids from g. 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.

TABLE XVII

Effect of DTT on the Half-Time of Autolytic Inactivation of Wild-Type and Disulfide Mutants of Subtilisin*

Enzyme_-DDT +DTT_- DTT/+DTT min

Wild-type 95 85 1.1 C22/C87 44 25 1.8 C24/C87 92 62 1.5 1 ' Purified enzymes were either treated or not treated with 25mM DTT and dialyzed with or without lOmM DTT in 2mM CaCl2, 50mM Tris (pH 7.5) for 14 hr. at 4®C. Enzyme concentrations were adjusted to 80μ1 aliquots were quenched on ice and assayed for residual activity. Half-times for autolytic inactivation were determined from semi-log plots of log1Q (residual activity) versus time. These plots were linear for over 90% of the inactivation.

TABLE XVIII

Effect of Mutations in Subtilisin on the Half-Time of Autolytic Inactivation at 58eC*

Enzvme min

Wild-type 120 C22 22 C24 120 C87 104 C22/C87 43 C24/C87 115 v ' Half-times for autolytic inactivation were determined for wild-type and mutant subtilisins as described in the legend to Table III. Unpurified and non-reduced enzymes were used directly from B. subtilis culture supernatants.

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. amvlolicrue-faciens 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 -5H 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 Acall fragment from pS4.5 which contains the 5' portion of the subtilisin gene and vector sequences, (2) the 200bp Avail fragment which contains the relevant 166 or 169 mutations from the respective 166 or 169 plasmids, and (3) the 2.2kb Avail 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.

TABLE XIX kcat Km WT 50 1.4xl0~4 A222 42 9.9xl0~4 K166 21 3.7xl0-5 K166/A222 29 2.0xl0-4 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 Xmal and treated with SI 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 BamHI and the approximately 4kb fragment containing the vector plus the 5' portion of the subtilisin gene through codon 167 was purified.

The pAl69 plasmid was digested with Kpnl and treated with DNA polymerase Klenow fragment plus 50 μΜ 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 BamHI 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 PvuII/Haell fragment containing the relevant 156, 166 and/or 169 mutations from the respective pl56, pl66 and/or pl69 double of single mutant plasmid, (2) the 550bp Haell/BamHI fragment containing the relevant 217 mutant from the respective p217 plasmid, and (3) the 3.9kb PvuII/BamHI 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, Gin, 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. amviol icruefaciens subtil is in 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 EcoRI-BamHI fragment of pBD64 (Gryczan, T., et al. (1980) J. Bacteriol., 141, 246-253) to give the recombinant plasmid pB0153. The unique EcoRI recognition sequence in pBD64 was eliminated by digestion with EcoRI 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 pE0154. The unique Aval recognition sequence in pB0154 was eliminated in a similar manner to yield pB0171.

pB0171 was digested with BamHI and PvuII 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. Ill, 97-120), to yield pB0172 which retains the unique BamHI site. To facilitate subcloning of subtilisin mutants, a unique and silent Kpnl 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 Kpnl+ plasmid was digested with EcoRI 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 BamHI. The 1.5 kb blunt EcoRI-BamHI fragment containing the entire subtilisin was ligated with the 5.8 kb Nrul-BamHI from pB0172 to yield pBO180. The ligation of the blunt Nrul end to the blunt EcoRI end recreated an EcoRI • site. Proceeding clockwise around pB0180 from the EcoRI site at the 5' end of the subtilisin gene is the unique BaroHI 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. m B. Construction of Random Mutagenesis Library

The 1.5 kb EcoRI-BamHI fragment containing the B. amyloliquefaciens subtilisin gene (Wells et al.f 1983) from pB0180 was cloned into M13mpll to give Ml3mpll 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 (Aval-) having the sequence

5'GAAAAAAGACCCTAGCGTCGCTTA ending at codon -11, was used to alter the unique Aval recognition sequence within the subtilisin gene. (The asterisk denotes the mismatches from the wild-type sequence and underlined is the altered Aval site.)

The 5' phosphorylated Aval primer (-320 pmol) and -40 pmol (-120yg) of uridine containing M13mpll SUBT template in 1.88 ml of 53 mM NaCl, 7.4 mM MgC12 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 100/iL containing 1 mM in all four deoxynucleotide triphosphates, and 20μ1 Klenow fragment (5 units/1). The extension reaction was stopped every 15 seconds over ten min by addition of 10μ1 0.25 M EDTA (pH 8) to 50pl 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 e-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 (_20^g), 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 37eC 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 ^-mercaptoethanol. Simultaneous restriction of each heteroduplex pool with KpnI. BamHI, and EcoRI confirmed that the extension reactions were nearly quantitative.

Heteroduplex DNA in each reaction mixture was methylated by incubation with 80 uM 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., 43-48). The number of independent transformants from each of the four transformations ranged from 0.4-2.0 x 10^. After growing out phage pools, RF DNA from each of the four transformations was isolated and purified by centrifugation through CsCl density gradients. Approximately 2ug of RF DNA from each of the four pools was digested with EcoRI, BamHI and Aval. The 1.5 kb EcoRI-BamHI fragment (i.e., Aval resistant) was purified on low gel temperature agarose and ligated into the 5.5 kb

EcoRI-BamHI vector fragment of pB0180. The total number of independent transformants from each α-thiodeoxynucleotide misincorporation plasmid library 4 ranged from 1.2-2.4 x 10 . The pool of plasmids from each of the four transformations was grown out in 200 ml LB media containing 12.5yg/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, 5yg of DNA produced approximately 5 2.5 x 10 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/5/ig/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 1 per well LB media plus 12.5Mg/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 subtil is in 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. T_, 1513) except that incubation with 2 mg/ml lysozyme proceeded for 5 min at 37°C to ensure cell lysis and an additional phenol/CHCl^ extraction was employed to remove contaminants. The 1.5 kb EcoRI-BamHI fragment containing the subtilisin gene was ligated into M13mpll 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 12.5ug/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 nmj_ c280* " 1*17 (Maturbara, H., et al. (1965), J. Biol. Chem, 240, 1125-1130).

Enzyme activity was measured with 200/zg/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-1; Del Mar, E.G., et al. (1979), Anal. Biochem.. 99, 316-320) . Alkaline autolytic stability studies were performed on purified enzymes (200pg/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 l. 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 Aval 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 l kb subtilisin gene fragment was assessed by multiple restriction digestion (not shown). For example, production of new Hinfl fragments identified when polymerase extension had proceeded past IlellO, Leu233, and Asp259 in the subtilisin gene.

Misincorporation of each dNTPos 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 dNTPas to the Aval 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 Aval restriction-selection against the wild-type template strand which contained a unique Aval site. The separate contribution of each of these enrichment procedures to the final mutagenesis frequency was not determined, except that prior to Aval restriction-selection roughly one-third of the segregated clones in each of the four pools still retained a wild-type Aval site within the subtilisin gene. After Aval restriction-selection greater than 98% of the plasmids lacked the wild-type Aval site.

The 1.5 kb EcoRI-BamHI subtilisin gene fragment that was resistant to Aval 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 dNTPas misincorporation reactions was estimated from the frequency that unique restriction sites were eliminated (Table XX) . The unique restriction sites chosen for this analysis, Clal, PvuII. and Kpnl, were distributed over the subtilisin gene starting at codons 35, 104, and 166, respectively. As a control, the mutagenesis frequency was determined at the PstI 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).

TABLE XX

is)

Mutagenesis frequency is estimated from the frequency for obtaining mutations that alter unique restriction sites within the mutagenized subtilisin gene (i.e., Clal. PvuII, or KpnH compared to mutation frequencies of the PstI site, that is outside the window of mutagenesis. ^ Plasmid DNA was from wild-type (none) or mutagenized by dNTPas misincorporation as described. ' ' Percentage of resistant clones was calculated from the fraction of clones obtained after three fold or greater over-digestion of the plasmid with the indicated restriction enzyme compared to a non-digested control. Restriction-resistant plasmid DNA from the first round was subjected to a second round of restriction-selection. The total represents the product of the fractions of resistant clones obtained from both rounds of selection and gives percentage of restriction-site mutant clones in the original starting pool. Frequencies were derived from counting at least 20 colonies and usually greater than 100. ^ Percent resistant clones was calculated by subtracting the percentage of restriction-resistant clones obtained for wild-type DNA (i.e., none) from that obtained for mutant DNA. ( p) ' ' This extrapolates from the frequency of mutation over each restriction site to the entire subtilisin gene (_1 kb) . This has been normalized to the number of possible bases (4-6 bp) within each restriction site that can be mutagenized by a given misincorporation event.

From this analysis, the average percentage of subtilisin genes containing mutations that result from dGTPas, dCTPas, or dTTPas misincorporation was estimated to be 90, 70, and 20 percent, respectively. These high mutagenesis frequencies were generally quite variable depending upon the dNTPas 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 dGTPas and dCTPas over dTTPas has been previously observed (Shortle, D. , et al. (1985), Genetics. 110. 539-555). Unlike the dGTPas, dCTPas, and dTTPas libraries the efficiency of mutagenesis for the dATPes 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 dATPas mutagenized subtilisin gene was subcloned from M13 into pB0180. Correcting for the vector background, we estimate the mutagenesis frequency around 20 percent in the dATPas misincorporation library. In a separate experiment (not shown), the mutagenesis efficiencies for dGTPas and dTTPas 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 athiodeoxy-nucleotide 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 dTTPas and dCTPas 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 dGTPas, dATPas, dTTPas, and dCTPas 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 EcoRI-Kpnl fragment of pB0180V107 into the 6.6 kb EcoRI-Kpnl fragment of pB0180R213), is more stable than either single mutant. The triple mutant, F50/V107/R213 (made by subcloning the 735 bp EcoRI-PvuII fragment of pF50 (Example 2) into the 6.8 kb EcoRI-PvuII 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).

TABLE XXI

Relationship between relative specific acitivity at pH S.6 or 10.8 and alkaline autolvtic stability

Alkaline

Relative specific activity autolysis half-time

Enzyme pH 8.6 pH 10.8 (min^b

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 ^ Relative specific activity was the average from triplicate activity determinations divided by the wild-type value at the same pH. The average specific activity of wild-type enzyme at pH 8.6 and 10.8 was 70μΐηο1β3/ιηΐη-ιι^ and 37Mmoles/min-mg, respectively. ^ Time to reach 50% activity was taken from Figs. 32 and 33. F. Random Cassette Mutagenesis of Residues 197 through 228

Plasmid pa222 (Wells, et al. (1985) Gene 34. 315-323) was digested with PstI and BamHI and the 0.4 kb Pstl/BamHI fragment (fragment 1, see Fig. 34) purified from a polyacrylamide gel by electroelution.

The 1.5 kb EcoRI/BamHI fragment from pS4.5 was cloned into M13mp9. Site directed mutagenesis was performed to create the A197 mutant and simultaneously insert a silent SstI site over codons 195-196. The mutant EcoRI/BamHI fragment was cloned back into pBS42. The pA197 plasmid was digested with BamHI and SstI and the 5.3 kb BamHI/SstI fragment (fragment 2) was purified from low melting agarose.

Complimentary oligonucleotides were synthesized to span the region from SstI (codons 195-196) to PstI (codons 228-230). These oligodeoxynucleotides were designed to (1) restore codon 197 to the wild type, (2) re-create a silent Kpnl site present in ρΔ222 at codons 219-220, (3) create a silent Smal site over codons 210-211, and (4) eliminate the PstI 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 4 represented 3.4 x 10 independent transformants. This plasmid pool was digested with PstI 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 150^1 of LB/12.5^g/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/^g/mL cmp plates and incubated overnight at 33eC (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 Na2C03, 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/20Mg/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 Smal restriction site (Fig. 35) and either ligating wild type sequence 3' to the Smal site to create the single C204 mutant or ligating wild type sequence 5' to the Smal 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.

TABLE XXII

Stability of subtilisin variants

Purified enzymes (200pg/mL) were incubated in 0.1M phosphate, pH 12 at 30°C for alkaline autolysis, or in 2mM CaCl2, 50mM MOPS, pH 7.0 at 62'C for thermal autolysis. At various times samples were assayed for residual enzyme activity. Inactivations were roughly pseudo-first order, and t 1/2 gives the time it took to reach 50% of the starting activity in two separate experiments. t 1/2 t 1/2 (alkaline (thermal autolysis) autolysis)

Exp. Exp. Exp. Exp.

Subtilisin variant #1 #2 #1 #2 wild type 30 25 20 23 F50/V107/R213 49 41 18 23 R204 35 32 24 27 C204 43 46 38 40 C204/R213 50 52 32 36 L204/R213 32 30 20 21 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 SstI and EcoRI and a 1.0 kb EcoRI/SstI fragment was isolated by electro-elution from polyacrylamide gel (fragment 1, see Fig. 35). C204/R213 was also digested with Smal and EcoRI 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 Smal 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 Smal-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.