AN IMPROVED CLEANING COMPOSITION

Abstract: Novel alpha-amylase mutants derived from the DNA sequences of naturally occurring or recombinant alpha-amylases are disclosed. The mutant alpha-amylases, in general, are obtained by in vitro modifications of a precursor DNA sequence encoding the naturally occurring or recombinant alpha-amylase to generate the substitution (replacement) or deletion of one or more oxidizable amino acid residues in the amino acid sequence of a precursor alpha-amylase. Such mutant alpha-amylases have altered oxidative stability and/or altered pH performance profiles and/or altered thermal stability as compared to the precursor. Also disclosed are detergent and starch liquefaction compositions comprising the mutant amylases, as well as methods of using the mutant amylases. More particularly preferred are mutant alpha-amylases from Bacillus licheniformis modified at MET197 or MET15 or at TRP138 residues or at equivalent residues of other alpha-amylases from other microbial sources (Bacillus, Aspergillus).

Description: AN IMPROVED CLEANING COMPOSITION _T_L«ld of th- Invention The present invention relates to novel alpha-amylase mutants having an amino acid sequence not found in nature, such mutants having an amino acid sequence wherein one or more amino acid residue(s) of a precursor alpha-amylase, specifically any oxidizable amino acid, have been substituted with a different amino acid. The mutant enzymes of the present invention exhibit altered stability/activity profiles including but not limited to altered oxidative stability, altered pH performance profile, altered specific activity and/or altered thermostability. Baghground of tht lavntion Alpha-amylases (alpha-l,4-glucan-4-glucanohydrolase, EC3.2.1.1) hydrolyze internal alpha-l,4-glucosidic linkages in starch largely at random, to produce smaller molecular weight malto-dextrins. Alpha-amylases are of considerable commercial value, being used in the initial stages (liquefaction) of starch processing; in alcohol production; as cleaning agents in detergent matrices; and in the textile industry for starch desizing. Alpha-amylases are produced by a wide variety of microorganisms including Bacillus and Aspergilluε , with most commercial amylases being produced from bacterial sources such as B. lichenifozmis, B. amyloliquefaciens, B. subtilis, or B. stearothermophilus . In recent years the preferred enzymes in commercial use have been those from B. licheniformis because of their heat stability and performance, at least at neutral and mildly alkaline pH's. Previously there have been studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases (Vihinen, M. et al . (1990) J. Biche . 107:267-272; Holm, L. et al. (1990) Protein Engineering 3:181-191; Takase, K. et al . (1992) Biochemica et Biophysica Acta, 1120:281-288; Matsui, I. et al. (1992) Febs Letters Vol. 310, No. 3, pp. 216-218); which residues are important for thermal stability (Suzuki, Y. et al. (1989) J. Biol. Chem. 264:18933-18938); and one group has used such methods to introduce mutations at various histidine residues in a B. licheniformis amylase, the rationale for making substitutions at histidine residues was that B. licheniformis amylase (known to be thermostable) when compared to other similar -Bacillus amylases, has an excess of histidines and, therefore, it was suggested that replacing a histidine could affect the thermostability of the enzyme (Declerck, N. et al. (1990) J. Biol. Chem. 265:15481-15488; FR 2 665 178-A1; Joyet, P. et al. (1992) Bio/Technology 10:1579-1583). It has been found that alpha-amylase is inactivated by hydrogen peroxide and other oxidants at pH's between 4 and 10.5 as described in the examples herein. Commercially, alpha-amylase enzymes can be used under dramatically different conditions such as both high and low pH conditions, depending on the commercial application. For example, alpha-amylases may be used in the liquefaction of starch, a process preferably performed at a low pH (pH <5.5). On the other hand, amylases may be used in commercial dish care or laundry detergents, which often contain oxidants such as bleach or peracids, and which are used in much more alkaline conditions. In order to alter the stability or activity profile of amylase enzymes under varying conditions, it has been found that selective replacement, substitution or deletion of oxidizable amino acids, such as a methionine, tryptophan, tyrosine, histidine or cysteine, results in an altered profile of the variant enzyme as compared to its precursor. Because currently commercially available amylases are not acceptable (stable) under various conditions, there is a need for an amylase having an altered stability and/or activity profile. This altered stability (oxidative, thermal or pH performance profile) can be achieved while maintaining adequate enzymatic activity, as compared to the wild-type or precursor enzyme. The characteristic affected by introducing such mutations may be a change in oxidative stability while maintaining thermal stability or vice versa . Additionally, the substitution of different amino acids for an oxidizable amino acids in the alpha- amylase precursor sequence or the deletion of one or more oxidizable amino acid(s) may result in altered enzymatic activity at a pH other than that which is considered optimal for the precursor alpha- amylase. In other words, the mutant enzymes of the present invention may also have altered pH performance profiles, which may be due to the enhanced oxidative stability of the enzyme. _B»™rm___ -u- pf thβ UlTlntlon The present invention relates to novel alpha-amylase mutants that are the expression product of a mutated DNA sequence encoding an alpha-amylase, the mutated DNA sequence being derived from a precursor alpha-amylase by the deletion or substitution (replacement) of one or more oxidizable amino acid. In one preferred embodiment of the present invention the mutant result from substituting a different amino acid for one or more methionine residue(s) in the precursor alpha-amylase. In another embodiment of the present invention the mutants comprise a substitution of one or more tryptophan residue alone or in combination with the substitution of one or more methionine residue in the precursor alpha-amylase. Such mutant alpha-amylases, in general, are obtained by in vi tro modification of a precursor DNA sequence encoding a naturally occurring or recombinant alpha-amylase to encode the substitution or deletion of one or more amino acid residues in a precursor amino acid sequence. Preferably the substitution or deletion of one or more amino acid in the amino acid sequence is due to the replacement or deletion of one or more methionine, tryptophan, cysteine, histidine or tyrosine residues in such sequence, most preferably the residue which is changed is a methionine residue. The oxidizable amino acid residues may be replaced by any of the other 20 naturally occurring amino acids. If the desired effect is to alter the oxidative stability of the precursor, the amino acid residue may be substituted with a non- oxidizable amino acid (such as alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, or valine) or another oxidizable amino acid (such as cysteine, methionine, tryptophan, tyrosine or histidine, listed in order of most easily oxidizable to less readily oxidizable) . Likewise, if the desired effect is to alter thermostability, any of the other 20 naturally occurring amino acids may be substituted (i.e., cysteine may be substituted for methionine) . Preferred mutants comprise the substitution of a methionine residue equivalent to any of the methionine residues found in B. licheniformis alpha-amylase (+8, +15, +197, +256, +304, +366 and +438) . Most preferably the methionine to be replaced is a methionine at a position equivalent to position +197 or +15 in B. licheniformis alpha-amylase. Preferred substitute amino acids to replace the methionine at position +197 are alanine (A) , isoleucine (I) , threonine (T) or cysteine (C) . The preferred substitute amino acids at position +15 are leucine (L) , threonine (T) , asparagine (N) , aspartate (D) , serine (S) , valine (V) and isoleucine (I) , although other substitute amino acids not specified above may be useful. Two specifically preferred mutants of the present invention are M197T and M15L. Another embodiment of this invention relates to mutants comprising the substitution of a tryptophan residue equivalent to any of the tryptophan residues found in B. licheniformis alpha-amylase (see Fig. 2) . Preferably the tryptophan to be replaced is at a position equivalent to +138 in B. licheniformis alpha-amylase. A mutation (substitution) at a tryptophan residue may be made alone or in combination with mutations at other oxidizable amino acid residues. Specifically, it may be advantageous to modify by substitution at least one tryptophan in combination with at least one methionine (for example, the double mutant +138 +197) . The alpha-amylase mutants of the present invention, in general, exhibit altered oxidative stability in the presence of hydrogen peroxide and other oxidants such as bleach or peracids, or, more specific, milder oxidants such as chloramine-T. Mutant enzymes having enhanced oxidative stability will be useful in extending the shelf life and bleach, perborate, percarbonate or peracid compatibility of amylases used in cleaning products. Similarly, reduced oxidative stability may be usefu.1 in industrial processes that require the rapid and efficient quenching * of enzymatic activity. The mutant enzymes of the present invention may also demonstrate a broadened pH performance profile whereby mutants such as M15L show stability for low pH starch liquefaction and mutants such as M197T show stability at high pH cleaning product conditions. The mutants of the present invention may also have altered thermal stability whereby the mutant may have enhanced stability at either high or low temperatures. It is understood that any change (increase or decrease) in the mutant's enzymatic characteristic(s) , as compared to its precursor, may be beneficial depending on the desired end use of the mutant alpha-amylase. In addition to starch processing and cleaning applications, variant amylases of the present invention may be used in any application in which known amylases are used, for example, variant amylases can be used in textile processing, food processing, etc. Specifically, it is contemplated that a variant enzyme such as M197C, which is easily inactivated by oxidation, would be useful in a process where it is desirable to completely remove amylase activity at the end of the process, for example, in frozen food processing applications. The preferred alpha-amylase mutants of the present invention are derived from a Bacillus strain such as B. licheniformis , B. amyloliquefacienε, and B. stearother ophiluε , and most preferably from Bacillus licheniformis . In another aspect of the present invention there is provided a novel form of the alpha-amylase normally produced by B. licheniformis . This novel form, designated as the A4 form, has an additional four alanine residues at the N-terminus of the secreted amylase. (Fig. 4b.) Derivatives or mutants of the A4 form of alpha-amylase are encompassed within the present invention. By derivatives or mutants of the A4 form, it is meant that the present invention comprises the A4 form alpha-amylase containing one or more additional mutations such as, for example, mutation (substitution, replacement or deletion) of one or more oxidizable amino acid(s) . In a composition embodiment of the present invention there are provided detergent compositions, liquid, gel or granular, comprising the alpha-amylase mutants described herein. Particularly preferred are detergent compositions comprising a +197 position mutant either alone or in combination with other enzymes such as endoglycosidases, cellulases, proteases, lipases or other amylase enzymes. Additionally, it is contemplated that the compositions of the present invention may include an alpha-amylase mutant having more than one site-specific mutation. In yet another composition embodiment of the present invention there are provided compositions useful in starch processing and particularly starch liquefaction. The starch liquefaction compositions of the present invention preferably comprise an alpha- amylase mutant having a substitution or deletion at position M15. Additionally, it is contemplated that such compositions may comprise additional components as known to those skilled in the art, including, for example, antioxidants, calcium, ions, etc. In a process aspect of the present invention there are provided methods for liquefying starch, and particularly granular starch slurries, from either a wet or dry milled process. Generally, in the first step of the starch degradation process, the starch slurry is gelatinized by heating at a relatively high temperature (up to about 110°C) . After the starch slurry is gelatinized it is liquefied and dextrinized using an alpha-amylase. The conditions for such liquefaction are described in commonly assigned US patent applications 07/785,624 and 07/785,623 and US Patent 5,180,669, the disclosure of which are incorporated herein by reference. The present method for liquefying starch comprises adding to a starch slurry an effective amount of an alpha-amylase of the present invention, alone or in combination with additional excipients such as an antioxidant, and reacting the slurry for an appropriate time and temperature to liquefy the starch. A further aspect of the present invention comprises the DNA encoding the mutant alpha-amylases of the present invention (including A4 form and mutants thereof) and expression vectors encoding the DNA as well as host cells transformed with such expression vectors. Bri«f D»«eriptlon of fchi Drawing■ Fig. 1 shows the DNA sequence of the gene for alpha-amylase from B. licheniformis (NCIB8061), Seq ID No 31, and deduced translation product as described in Gray, G. et al. (1986) J. Bacter. 166:635- 643. Fig. 2 shows the amino acid sequence of the mature alpha-amylase enzyme from B. licheniformis (NCIB8061) , Seq ID No 32. Fig. 3 shows an alignment of primary structures of Bacillus alpha- amylases. The B . licheniformis amylase (Am-Lich) , Seq ID No 33, is described by Gray, G. et al. (1986) J. Bact. 166:635-643; the B. amyl oliquefacienε amylase (Am-Amylo) , Seq ID No 34, is described by Takkinen, K. et al. (1983) J. Biol. Chem. 258:1007-1013; and the B. stearothermophilus (Am-Stearo) , Seq ID No 35, is described by Ihara, H. et al. (1985) J. Bioche . 98:95-103. Fig. 4a shows the amino acid sequence of the mature alpha-amylase variant M197T, Seq ID No 36. Fig. 4b shows the amino acid sequence of the A4 form of alpha- amylase from B. licheniformis NCIB8061, Seq ID No 37. Numbering is from the N-terminus, starting with the four additional alanines. Fig. 5 shows plasmid pA4BL wherein BLAA refers to B. licheniformiε alpha-amylase gene, Pstl to Sstl; Amp* refers to the ampicillin- resistant gene from pBR322; and CAT refers to the Chloramphenicol- resistant gene from pC194. Fig. 6 shows the signal sequence-mature protein junctions for B. licheniformiε (Seq ID No 38), B. εubtilis (Seq ID No 39), B. licheniformiε in pA4BL (Seq ID No 40) and B. licheniformiε in pBLapr (Seq ID No 41) . Fig. 7a shows inactivation of certain alpha-amylases (Spezyme® AA20 and M197L (A4 form) with 0.88M H 2 0 2 at pH 5.0, 25°C. Fig. 7b shows inactivation of certain alpha-amylases (Spezyme® AA20, M197T) with 0.88M H 2 0 2 at pH 10.0, 25°C. Fig. 7c shows inactivation of certain alpha-amylases (Spezyme® AA20, M15L) with 0.88M H 2 0 2 at pH 5.0, 25°C. Fig. 8 shows a schematic for the production of M197X cassette mutants. Fig. 9 shows expression of M197X variants. Fig. 10 shows thermal stability of M197X variants at pH 5.0, 5mM CaCl 2 at 95°C for 5 mins. Figs. 11a and lib show inactivation of certain amylases in automatic dish care detergents. Fig. 11a shows the stability of certain amylases in Cascade™ (a commercially available dish care product) at 65°C in the presence or absence of starch. Fig. lib shows the stability of certain amylases in Sunlight™ (a commercially available dish care product) at 65°C in the presence or absence of starch. Fig. 12 shows a schematic for the production of M15X cassette mutants. Fig. 13 shows expression of M15X variants. Fig. 14 shows specific activity of M15X variants on soluble starch. Fig. 15 shows heat stability of M15X variants at 90°C, pH 5.0, 5mM CaCl 2 , 5 ins. Fig. 16 shows specific activity on starch and soluble substrate, and performance in jet liquefaction at pH 5.5, of M15 variants as a function of percent activity of B. licheniformiε wild-type. Fig. 17 shows the inactivation of B. licheniformiε alpha-amylase (AA20 at 0.65 g/ml) with chloramine-T at pH 8.0 as compared to variants M197A (1.7 mg/ml) and M197L (1.7 mg/ml). Fig. 18 shows the inactivation of B. licheniformiε alpha-amylase (AA20 at 0.22 mg/ml) with chloramine-T at pH 4.0 as compared to variants M197A (4.3 mg/ml) and M197L (0.53 mg/ml). Fig. 19 shows the reaction of B. licheniformiε alpha r amylase (AA20 at 0.75 mg/ml) with chloramine-T at pH 5.0 as compared to double variants M197T/W138F (0.64 mg/ml) and M197T/W138Y (0.60 mg/ml). Fig. 20 shows the stability testing results of various alpha-amylase multiple mutants incorporated in automatic dish detergent (ADD) formulations at temperatures from room temperature increased to 65°C. Fig. 21 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at room temperature over 0-30 days, as determined by percent activity remaining over time. Fig. 22 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at 38°C (100°F) with 80% relative humidity over 0-30 days. Detailed Description of the Invtntlon It is believed that amylases used in starch liquefaction may be subject to some form of inactivation due to some activity present in the starch slurry (see commonly owned US applications 07/785,624 and 07/785,623 and US Patent 5,180,669, issued January 19, 1993, incorporated herein by reference) . Furthermore, use of an amylase in the presence of oxidants, such as in bleach- or peracid- containing detergents, may result in partial or complete inactivation of the amylase. Therefore, the present invention focuses on altering the oxidative sensitivity of amylases. The mutant enzymes of the present invention may also have an altered pH profile and/or altered thermal stability which may be due to the enhanced oxidative stability of the enzyme at low or high pH's. Alpha-amylase as used herein includes naturally occurring amylases as well as recombinant amylases. Preferred amylases in the present invention are alpha-amylases derived from B. licheniformiε or B. εtearothermophiluε, including the A4 form of alpha-amylase derived from B. licheniformiε as described herein, as well as fungal alpha- amylases such as those derived from Aεpergilluε (i.e., A. oryzae and A . niger) . Recombinant alpha-amylases refers to an alpha-amylase in which the DNA sequence encoding the naturally occurring alpha-amylase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the alpha-amylase sequence. Suitable modification methods are disclosed herein, and also in commonly owned US Patents 4,760,025 and 5,185,258, the disclosure of which are incorporated herein by reference. Homologies have been found between almost all endo-amylases sequenced to date, ranging from plants, mammals, and bacteria (Nakajima, R.T. et al. (1986) Appl. Microbiol. Biotechnol. 23:355- 360; Rogers, J.C. (1985) Biochem. Biophys. Res. Commun. 128:470- 476) . There are four areas of particularly high homology in certain Bacilluε amylases, as shown in Fig. 3, wherein the underlined sections designate the areas of high homology. Further, sequence alignments have been used to map the relationship between Bacilluε endo-amylases (Feng, D.F. and Doolittle, R.F. (1987) J. Molec. Evol. 35:351-360). The relative sequence homology between B. εtearothermophiluε and B. licheniformiε amylase is about 66%, as determined by Holm, L. et al. (1990) Protein Engineering _ (3) pp. 181-191. The sequence homology between B. licheniformiε and B. a yloliquefacienε amylases is about 81%, as per Holm, L. et al., εupra . While sequence homology is important, it is generally recognized that structural homology is also important in comparing amylases or other enzymes. For example, structural homology between fungal amylases and bacterial (Bacilluε) amylase have been suggested and, therefore, fungal amylases are encompassed within the present invention. An alpha-amylase mutant has an amino acid sequence which is derived from the amino acid sequence of a precursor alpha-amylase. The precursor alpha-amylases include naturally occurring alpha-amylases and recombinant alpha-amylases (as defined) . The amino acid sequence of the alpha-amylase mutant is derived from the precursor alpha-amylase 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 alpha-amylase rather than manipulation of the precursor alpha-amylase enzyme per εe. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in commonly owned US patent 4,760,025 and 5,185,258. Specific residues corresponding to positions M197, M15 and W138 of Bacilluε licheniformiε alpha-amylase are identified herein for substitution or deletion, as are all methionine, histidine, tryptophan, cysteine and tyrosine positions. The amino acid position number (i.e., +197) refers to the number assigned to the mature Bacilluε licheniformiε alpha-amylase sequence presented in Fig. 2. The invention, however, is not limited to the mutation of this particular mature alpha-amylase (B. licheniformiε) but extends to precursor alpha-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in B. licheniformiε alpha-amylase. A residue (amino acid) of a precursor alpha-amylase is equivalent to a residue of B. licheniformiε alpha-amylase if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. licheniformiε alpha-amylase (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally) . In order to establish homology to primary structure, the amino acid sequence of a precursor alpha-amylase is directly compared to the B. licheniformiε alpha-amylase primary sequence and particularly to a set of residues known to be invariant to all alpha-amylases for which sequence is known, as seen in Fig. 3. It is possible also to determine equivalent residues by tertiary structure: crystal structures have been reported for porcine pancreatic alpha-amylase (Buisson, G. et al. (1987) EMBO J.6:3909-3916) ; Taka-amylase A from Aεpergilluε oryzae (Matsuura, Y. et al. (1984) J. Biochem. (Tokyo) 95:697-702); and an acid alpha-amylase from A . niger (Boel, E. et al. (1990) Biochemistry 29:6244-6249), with the former two structures being similar. There are no published structures for Bacilluε alpha-amylases, although there are predicted to be common super-secondary structures between glucanases (MacGregor, E.A. & Svensson, B. (1989) Biochem. J. 259:145-152) and a structure for the B. εtearothermophiluε enzyme has been modeled on that of Taka- amylase A (Holm, L. et al. (1990) Protein Engineering 3:181-191). The four highly conserved regions shown in Fig. 3 contain many residues thought to be part of the active-site (Matsuura, Y. et al. (1984) J. Biochem. (Tokyo) 95:697-702; Buisson, G. et al. (1987) EMBO J. 6:3909-3916; Vihinen, M. et al. (1990) J. Biochem. 107:267- 272) including, in the licheniformiε numbering, Hisl05; Arg229; Asp231; His235; Glu261 and Asp328. Expression vector as used herein 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 may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable RNA ribosome-binding sites, and sequences which control termination of transcription and translation. A preferred promoter is the B. εubtiliε aprE promoter. 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. Host strains (or cells) useful in the present invention generally are procaryotic or eucaryotic hosts and include any transformable microorganism in which the expression of alpha-amylase can be achieved. Specifically, host strains of the same species or genus from which the alpha-amylase is derived are suitable, such as a Bacilluε strain. Preferably an alpha-amylase negative Bacilluε strain (genes deleted) and/or an alpha-amylase and protease deleted Bacilluε strain such as Bacilluε εubtilis strain BG2473 ( amyΣ, Lapr, npr) is used. Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the alpha-amylase and its variants (mutants) or expressing the desired alpha-amylase. Preferably the mutants of the present invention are secreted into the culture medium during fermentation. Any suitable signal sequence, such as the aprE signal peptide, can be used to achieve secretion. Many of the alpha-amylase mutants of the present invention are useful in formulating various detergent compositions, particularly certain dish care cleaning compositions, especially those cleaning compositions containing known oxidants. Alpha-amylase mutants of the invention can be formulated into known powdered, liquid or gel detergents having pH between 6.5 to 12.0. Suitable granular composition may be made as described in commonly owned US patent applications 07/429,881, 07/533,721 and 07/957,973, all of which are incorporated herein by reference. These detergent cleaning compositions can also contain other enzymes, such as known proteases, lipases, cellulases, endoglycosidases or other amylases, as well as builders, stabilizers or other excipients known to those skilled in the art. These enzymes can be present as co-granules or as blended mixes or in any other manner known to those skilled in the art. Furthermore, it is contemplated by the present invention that multiple mutants may be useful in cleaning or other applications. For example, a mutant enzyme having changes at both +15 and +197 may exhibit enhanced performance useful in a cleaning product or a multiple mutant comprising changes at +197 and +138 may have improved performance. Specifically preferred mutant enzymes for use in cleaning products, and particularly dish care formulations, include but are not limited to M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T; and M15T/W138Y/M197T. Another embodiment of the present invention comprises the combination of the mutant alpha-amylase enzymes described herein in combination with other enzymes (i.e., proteases, lipases, cellulases, etc.), and preferably oxidatively stable proteases. Suitable oxidatively stable proteases include genetically engineered proteases such as those described in US Re 34606, incorporated herein by reference, as well as commercially available enzymes such as DURAZYM (Novo Nordisk), MAXAPEM (Gist-brocades) and PURAFECT OXP (Genencor International, Inc.). Suitable methods for making such protease mutants (oxidatively stable proteases), and particularly such mutants having a substitution for the methionine at a position equivalent to M222 in B. amyloliquefaci ens, are described in US Re 34606. Suitable methods for determining "equivalent" positions in other subtilisins are provided in Re 34606, EP 257,446 and USSN 212,291, which are incorporated herein by reference. As described previously, alpha-amylase mutants of the present invention may also be useful in the liquefaction of starch. Starch liquefaction, particularly granular starch slurry liquefaction, is typically carried out at near neutral pH's and high temperatures. As described in commonly owned US applications 07/788,624 and 07/785,623 and US Patent 5,180,669, it appears that an oxidizing agent or inactivating agent of some sort is also present in typical liquefaction processes, which may affect the enzyme activity; thus, in these related patent applications an antioxidant is added to the process to protect the enzyme. Based on the conditions of a preferred liquefaction process, as described in commonly owned US applications 07/788,624 and 07/785,623 and US Patent 5,180,669, namely low pH, high temperature and potential oxidation conditions, preferred mutants of the present invention for use in liquefaction processes comprise mutants exhibiting altered pH performance profiles (i.e., low pH profile, pH <6 and preferably pH <5.5), and/or altered thermal stability (i.e., high temperature, about 90°-110°C) , and/or altered oxidative stability (i.e., enhanced oxidative stability). Thus, an improved method for liquefying starch is taught by the present invention, the method comprising liquefying a granular starch slurry from either a wet or dry milling process at a pH from about 4 to 6 by adding an effective amount of an alpha-amylase mutant of the present invention to the starch slurry; optionally adding an effective amount of an antioxidant or other excipient to the slurry; and reacting the slurry for an appropriate time and temperature to liquefy the starch. The following is presented by way of example and is not to be construed as a limitation to the scope of the claims. Abbreviations used herein, particularly three letter or one letter notations for amino acids are described in Dale, J.W., Molecular Genetics of Bacteria, John Wiley & Sons, (1989) Appendix B. Bwtrintntt 1 Example 1 Substitutions for the Methionine Residues in B . l i cheni formiε Alpha-Aπrvlase The alpha-amylase gene (Fig. 1) was cloned from B. licheniformiε NCIB8061 obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (Gray, G. et al. (1986) J. Bacteriology 166:635-643). The 1.72kb Pstl-Sstl fragment, encoding the last three residues of the signal sequence; the entire mature protein and the terminator region was subcloned into M13MP18. A synthetic terminator was added between the Bell and Sstl sites using a synthetic oligonucleotide cassette of the form: 5' GATCAAAACATAAAAAACCGGCCTTGGCCCCGCCGGTTTTTTATTATTTTTGAGCT 3 ' 3 ' TTTTGTATTTTTTGGCCGGAACCGGGGCGGCCAAAAAATAATAAAAAC 5' Seq ID No 1 designed to contain the B. amyloliquefacienε subtilisin transcriptional terminator (Wells et al. (1983) Nucleic Acid Research 11:7911-7925). Site-directed mutagenesis by oligonucleotides used essentially the protocol of Zoller, M. et al. (1983) Meth. Enzymol. 100:468-500: briefly, 5'-phosphorylated oligonucleotide primers were used to introduce the desired mutations on the M13 single-stranded DNA template using the oligonucleotides listed in Table I to substitute for each of the seven methionines found in B. licheniformiε alpha- amylase. Each utagenic oligonucleotide also introduced a restriction endonuclease site to use as a screen for the linked mutation. TABLE I Mutaαenic Ol αonuc__ent.ides for the Subs itu ion of he Methionine Residues in B. licheniformis Alcha-Amvlase M8A 5'-T GGG ACG CTG OCG CAG TAC TTT GAA TGG T-3 ' Seq ID No 2 Scal+ M15L 5 '-TG ATG CAG TAC TTT GAA TGG TAC CTG CCC AAT GA-3 ' Seq ID No 3 Scal+ Kpnl+ M197L 5 '-GAT TAT TTG TTG TAT GCC GAT ATC GAC TAT GAC CAT-3 ' Seq ID No 4 EcoRV+ M256A 5'-CG GGG AAG GAQ Qcc TTT ACG GTA GCT-3 ' Seq ID No 5 Stul+ M304L 5"-GC GGC TAT GAC TTA AGG AAA TTG C-3 ' Seq ID No 6 AfIII+ M366A 5'-C TAC GGG GAT OCA TAC GGG ACG A-3" Seq ID No 7 Nsil+ M366Y 5 '-C TAC GGG GAT TAC TAC GGG ACC AAo GGA GAC TCC C-3 ' Seq ID No 8 Styl+ M438A 5'-CC GGT GGG GCC AAG CGQ QCC TAT GTT GGC CGG CAA A-3" Seq ID No 9 Sfil+ Bold letter indicate base changes introduced by oligonucleotide. Codon changes indicated in the form M8A, where methionine (M) at position +8 has been changed to alanine (A) . Underlining indicates restriction endonuclease site introduced by oligonucleotide. The heteroduplex was used to transfect E. coli mutL cells (Kramer et al. (1984) Cell 38:879) and, after plaque-purification, clones were analyzed by restriction analysis of the RFl's. Positives were confirmed by dideoxy sequencing (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and the Pstl-Sstl fragments for each subclone ' d into an E. coli vector, plasmid pA4BL. Plasmid τ>A4BL Following the methods described in US application 860,468 (Power et al.), which is incorporated herein by reference, a silent Pstl site was introduced at codon +1 (the first amino-acid following the signal cleavage site) of the aprE gene from pS168-l (Stahl, M.L. and Ferrari, E. (1984) J. Bacter. 158:411-418) . The aprE promoter and signal peptide region was then cloned out of a pJHIOl plasmid (Ferrari, F.A. et al. (1983) J. Bacter. 154:1513-1515) as a Hindlll- Pstl fragment and subcloned into the pUClβ-derived plasmid JM102 (Ferrari, E. and Hoch, J.A. (1989) Bacillus, ed. C.R. Harwood, Plenum Pub., pp. 57-72). Addition of the Pstl-Sstl fragment from B. licheniformiε alpha-amylase gave pA4BL (Fig. 5) having the resulting aprE signal peptide-amylase junction as shown in Fig. 6. Transformation Into B . subti l i ε pA4BL is a plasmid able to replicate in E. coli and integrate into the B. εubtiliε chromosome. Plasmids containing different variants were transformed into B. εubtiliε (Anagnostopoulos, C. and Spizizen, J. (1961) J. Bacter. 81:741-746) and integrated into the chromosome at the aprE locus by a Campbell-type mechanism (Young, M. (1984) J. Gen. Microbiol. 130:1613-1621). The Bacilluε subtilis strain BG2473 was a derivative of 1168 which had been deleted for amylase (Δa yE) and two proteases ( apr, npr) (Stahl, M.L. and Ferrari, E., J. Bacter. 158:411-418 and US Patent 5,264,366, incorporated herein by reference). After transformation the sacϋ32 (Hy) (Henner, D.J. et al. (1988) J. Bacter. 170:296-300) mutation was introduced by PBS-1 mediated transduction (Hoch, J.A. (1983) 154:1513-1515). N-terminal analysis of the amylase expressed from pA4BL in B. εubtiliε showed it to be processed having four extra alanines at the N-terminus of the secreted amylase protein ("A4 form"). These extra residues had no significant, deleterious effect on the activity or thermal stability of the A4 form and in some applications may enhance performance. In subsequent eaqperiments the correctly processed forms of the licheniformiε amylase and the variant M197T were made from a very similar construction (see Fig. 6) . Specifically, the 5' end of the A4 construction was subcloned on an EcoRI-Sstll fragment, from pA4BL (Fig. 5) into M13BM20 (Boehringer Mannheim) in order to obtain a coding-strand template for the mutagenic oligonucleotide below: 5"-CAT CAG CGT CCC ATT AAG ATT TGC AGC CTG CGC AGA CAT GTT GCT-3 ' Seq ID No 10 This primer eliminated the codons for the extra four N-terminal alanines, correct forms being screened for by the absence of the Pstl site. Subcloning the EcoRI-Sstll fragment back into the pA4BL vector (Fig. 5) gave plasmid pBLapr. The M197T substitution could then be moved, on a Sstll-Sstl fragment, out of pA4BL (M197T) into the complementary pBLapr vector to give plasmid pBLapr (M197T) . N- terminal analysis of the amylase expressed from pBLapr in B. εubtiliε showed it to be processed with the same N-terminus found in B. licheniformiε alpha-amylase. Example 2 Oxidative Sensitivity of Methionine Variants B. licheniformiε alpha-amylase, such as Spezyme® AA20 (commercially available from Genencor International, Inc.), is inactivated rapidly in the presence of hydrogen peroxide (Fig. 7) . Various methionine variants were expressed in shake-flask cultures of B. subtilis and the crude supernatants purified by ammonium sulphate cuts. The amylase was precipitated from a 20% saturated ammonium sulphate supernatant by raising the ammonium sulphate to 70% saturated, and then resuspended. The variants were then exposed to 0.88M hydrogen peroxide at pH 5.0, at 25°C. Variants at six of the methionine positions in B. licheniformiε alpha-amylase were still subject to oxidation by peroxide while the substitution at position +197 (M197L) showed resistance to peroxide oxidation. (See Fig. 7.) However, subsequent analysis described in further detail below showed that while a variant may be susceptible to oxidation at pH 5.0, 25°C, it may exhibit altered/enhanced properties under different conditions (i.e., liquefaction). Example 3 Construction of All Possible Variants at Position 1 Q7 All of the M197 variants (M197X) were produced in the A4 form by cassette mutagenesis, as outlined in Fig. 8: 1) Site directed mutagenesis (via primer extension in M13) was used to make M197A using the mutagenic oligonucleotide below: M197A 5'-GAT TAT TTG OCG TAT GCC GAT ATC GAC TAT GAC CAT-3 ' EcoRV+ Clal- Seq ID No 11 which also inserted an EcoRV site (codons 200-201) to replace the Clal site (codons 201-202) . 2) Then primer LAAM12 (Table II) was used to introduce another silent restriction site (BstBI) over codons 186-188. 3) The resultant M197A (BstBI+, EcoRV+) variant was then subcloned (Pstl-Sstl fragment) into plasmid pA4BL and the resultant plasmid digested with BstBI and EcoRV and the large vector-containing fragment isolated by electroelution from agarose gel. 4) Synthetic primers LAAM14-30 (Table II) were each annealed with the largely complementary common primer LAAM13 (Table II) . The resulting cassettes encoded for all the remaining naturally occurring amino acids at position +197 and were ligated, individually, into the vector fragment prepared above. TABLE 11 Synthetic Oligonucleotides Used for Cassette Mutagenesis to Produce M197X Variants LAAJM12 GG GAA GTT TCG AAT GAA AAC G Seq ID No 12 IΛAM13 Xl97bs Seq ID No 13 (EcoRV) STC GGC ATA TG CAT ATA ATC ATA GTT GCC GTT TTC ATT (BstBI) I_ΛAM14 1197 Seq ID No 14 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG &TC TAT GCC GAC (EcoRV- XAAM15 F197 Seq ID No 15 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG H TAT GCC GAC (EcoRV- r* IAAM16 V197 Seq ID No 16 o (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG SH TAT GCC GAC (EcoRV- LAAM17 S197 Seq ID No 17 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG &QC TAT GCC GAC (EcoRV- XAAM18 P197 Seq ID No 18 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG CC1 TAT GCC GAC (EcoRV- IAΛM19 T197 Seq ID No 19 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG ΔCΔ TAT GCC GAC (EcoRV- UUU420 Y197 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG T&C TAT GCC GAC (EcoRV- IAAM21 H197 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG Δ TAT GCC GAC (EcoRV- IAAM22 G197 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG GJ3C TAT GCC GAC (EcoRV- CG AAT GAA AAC GGC AAC TAT GAT TAT TTG CM TAT GCC GAC CG AAT GAA AAC GGC AAC TAT GAT TAT TTG AAC TAT GCC GAC CG AAT GAA AAC GGC AAC TAT GAT TAT TTG & TAT GCC GAC CG AAT GAA AAC GGC AAC TAT GAT TAT TTG Q&Σ. TAT GCC GAC CG AAT GAA AAC GGC AAC TAT GAT TAT TTG G TAT GCC GAC CG AAT GAA AAC GGC AAC TAT GAT TAT TTG TGJT TAT GCC GAC CG AAT GAA AAC GGC AAC TAT GAT TAT TTG ΪQQ TAT GCC GAC r CG AAT GAA AAC GGC AAC TAT GAT TAT TTG &S& TAT GCC GAC The cassettes were designed to destroy the EcoRV site upon ligation, thus plasmids from E. coli transformants were screened for loss of this unique site. In addition, the common bottom strand of the cassette contained a frame-shift and encoded a Nsil site, thus transformants derived from this strand could be eliminated by screening for the presence of the unique Nsil site and would not be expected, in any case, to lead to expression of active amylase. Positives by restriction analysis were confirmed by sequencing and transformed in B. subtilis for expression in shake-flask cultures (Fig. 9). The specific activity of certain of the M197X mutants was then determined using a soluble substrate assay. The data generated using the following assay methods are presented below in Table III. Soluble Substrate Assay: A rate assay was developed based on an. end-point assay kit supplied by Megazyme (Aust.) Pty. Ltd.: Each vial of substrate (p.-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10ml of sterile water, followed by a 1 to 4 dilution in assay buffer (50mM maleate buffer, pH 6.7, 5mM calcium chloride, 0.002% Tween20) . Assays were performed by adding lOut of amylase to 790u< of the substrate in a cuvette at 25°C. Rates of hydrolysis were measured as the rate of change of absorbance at 410nm, • after a delay of 75 seconds. The assay was linear up to rates of 0.4 absorption units/min. The amylase protein concentration was measured using the standard Bio-Rad assay (Bio-Rad Laboratories) based on the method of Bradford, M. (1976) Anal. Biochem. 72:248) using bovine serum albumin standards. Starch Hydrolysis Assay: The standard method for assaying the alpha-amylase activity of Spezyme® AA20 was used. This method is described in detail in Example 1 of USSN 07/785,624, incorporated herein by reference. Native starch forms a blue color with iodine but fails to do so when it is hydrolyzed into shorter dextrin molecules. The substrate is soluble Lintner starch 5gm/liter in phosphate buffer, pH 6.2 (42.5gm/liter potassium dihydrogen phosphate, 3.16gm/liter sodium hydroxide). The sample is added in 25mM calcium chloride and activity is measured as the time taken to give a negative iodine test upon incubation at 30°C. Activity is recorded in liquefons per gram or ml (LU) calculated according to the formula: LU/ml or LU/g = 570 x D V x t Where LU=liquefon unit V=volume of sample (5ml) t=dextrinization time (minutes) D=dilution factor=dilution volume/ml or g of added enzyme. Variant M15L made as per the prior examples did not show increased amylase activity (Table III) and was still inactivated by hydrogen peroxide (Fig. 7) . It did, however, show significantly increased performance in jet-liquefaction of starch, especially at low pH as shown in Table IV below. Starch liquefaction was typically performed using a Hydroheater M 103-M steam jet equipped with a 2.5 liter delay coil behind the mixing chamber and a terminal back pressure valve. Starch was fed to the jet by a Moyno pump and steam was supplied by a 150 psi steam line, reduced to 90-100 psi. Temperature probes were installed just after the Hydroheater jet and just before the back pressure valve. Starch slurry was obtained from a corn wet miller and used within two days. The starch was diluted to the desired solids level with deionized water and the pH of the starch was adjusted with 2% NaOH or saturated Na 2 C0 3 . Typical liquefaction conditions were: Starch 32%-35% solids Calcium 40-50 ppm (30 pp added) pH 5.0-6.0 Alpha-amylase 12-14 LU/g starch dry basis Starch was introduced into the jet at about 350 ml/min. The jet temperature was held at 105°-107°C. Samples of starch were transferred from the jet cooker to a 95°C second stage liquefaction and held for 90 minutes. The degree of starch liquefaction was measured immediately after the second stage liquefaction by determining the dextrose equivalence (DE) of the sample and by testing for the presence of raw starch, both according to the methods described in the Standard Analytical Methods of the Member Companies of the Corn Refiners Association. Inc.. sixth edition. Starch, when treated generally under the conditions given above and at pH 6.0, will yield a liquefied starch with a DE of about 10 and with no raw starch. Results of starch liquefaction tests using mutants of the present invention are provided in Table IV. TABLE IV Performance of Variants M15L (A4 form, and M15L in Starch Liquefaction fiϋ DE after 90 Mi s. Spezy eΦ AA20 5.9 9.9 M15L (A4 form) 5.9 10.4 SpezymeΦ AA20 5.2 1.2 M15L (A4 form) 5.2 2.2 Spezyme® AA20 5.9 9.3* M15L 5.9 11.3* SpezymeΦ AA20 5.5 3.25** M15L 5.5 6.7** Spezyme® AA20 5.2 0.7 I"** M15L 5.2 3.65 average of three experiments average of two experiments Example 5 Construction of M15X Variants Following generally the processes described in Example 3 above, all variants at M15 (M15X) were produced in native B. licheniformiε by cassette mutagenesis, as outlined in Fig. 12: 1) Site directed mutagenesis (via primer extension in M13) was used to introduce unique restriction sites flanking the M15 codon to facilitate insertion of a mutagenesis cassette. Specifically, a BstBI site at codons 11-13 and a Mscl site at codons 18-20 were introduced using the two oligonucleotides shown below: M15XBstBl 5'-G ATG CAG TAT TTC GAA CTGG TAT A-3' BstBI Seq ID No 48 M15XMSC1 5'-TG CCC AAT GAT GGC CAA CAT TGG AAG-3 ' Mscl Seq ID No 49 2) The vector for M15X cassette mutagenesis was then constructed by subcloning the Sfil-Sstll fragment from the mutagenized amylase (BstBl+, Mscl+) into plasmid pBLapr. The resulting plasmid was then digested with BstBI and Mscl and the large vector fragment isolated by electroelution from a polyacrylamide gel. 3) Mutagenesis cassettes were created as with the M197X variants. Synthetic oligomers, each encoding a substitution at codon 15, were annealed to a common bottom primer. Upon proper ligation of the cassette to the vector, the Mscl is destroyed allowing for screening of positive transformants by loss of this site. The bottom primer contains an unique SnaBl site allowing for the transformants derived from the bottom strand to be eliminated by screening for the SnaBl site. This primer also contains a frameshift which would also eliminate amylase expression for the mutants derived from the common bottom strand. The synthetic cassettes are listed in Table V and the general cassette mutagenesis strategy is illustrated in Figure 12. TABLE V Synthetic Oligonucleotides Used for Cassette Mutagenesis to Produce M15X Variants M15A (BstBI) C GAA TGG TAT fid CCC AAT GAC GG (Mscl) Seq ID No 50 M15R (BstBI) C GAA TGG TAT £££ CCC AAT GAC GG (Mscl) Seq ID No 51 M15N (BstBI) C GAA TGG TAT AAI CCC AAT GAC GG (Mscl) Seq ID No 52 M15D (BstBI) C GAA TGG TAT S T CCC AAT GAC GG (Mscl) Seq ID No 53 M15H (BstBI) C GAA TGG TAT £A£ CCC AAT GAC GG (Mscl) Seq ID No 54 M15K (BstBI) C GAA TGG TAT AAA CCC AAT GAC GG (Mscl) Seq ID No 55 M15P (BstBI) C GAA TGG TAT £C£ CCC AAT GAC GG (Mscl) Seq ID No 56 M15S (BstBI) C GAA TGG TAT TCI CCC AAT GAC GG (Mscl) Seq ID No 57 M15T (BstBI) C GAA TGG TAC ACT. CCC AAT GAC GG (Mscl) Seq ID No 58 M15V (BstBI) C GAA TGG TAT SIT CCC AAT GAC GG (Mscl) Seq ID No 59 M15C (BstBI) C GAA TGG TAT 1ST CCC AAT GAC GG (Mscl) Seq ID No 60 M15Q (BstBI) C GAA TGG TAT £AA CCC AAT GAC GG (Mscl) Seq ID No 61 M15E (BstBI) C GAA TGG TAT S CCC AAT GAC GG (Mscl) Seq ID No 62 M15G (BstBI) C GAA TGG TAT fi£I CCC AAT GAC GG (Mscl) Seq ID No 63 M15I (BstBI) C GAA TGG TAT All CCC AAT GAC GG (Mscl) Seq ID No 64 M15F (BstBI) C GAA TGG TAT 332 CCC AAT GAC GG (Mscl) Seq ID No 65 M15W (BstBI) C GAA TGG TAC 2S£ CCC AAT GAC GG (Mscl) Seq ID No 66 M15Y (BstBI) C GAA TGG TAT TAT CCC AAT GAC GG (Mscl) Seq ID No 67 M15X (Mscl) CC GTC ATT GGG ACT ACG TAC CAT T (BstBI) Seq ID No 68 (bottom strand) Underline indicates codon changes at amino acid position 15. Conservative substitutions were made in some cases to prevent introduction of new restriction sites. Example 6 Bench Liquefaction with M15X Variant? Eleven alpha-amylase variants with substitutions for M15 made as per Example 5 were assayed for activity, as compared to SpezymeΦ AA20 (commercially available from Genencor International, Inc.) in liquefaction at pH 5.5 using a bench liquefaction system. The bench scale liquefaction system consisted of a stainless steel coil (0.25 inch diameter, approximately 350 ml volume) equipped with a 7 inch long static mixing element approximately 12 inches from the anterior end and a 30 psi back pressure valve at the posterior end. The coil, except for each end, was immersed in a glycerol-water bath equipped with thermostatically controlled heating elements that maintained the bath at 105-106°C. Starch slurry containing enzyme, maintained in suspension by stirring, was introduced into the reaction coil by a piston driven metering pump at about 70 ml/min. The starch was recovered from the end of the coil and was transferred to the secondary hold (95°C for 90 minutes) . Immediately after the secondary hold, the DE of the liquefied starch was determined, as described in Example 4. The results are shown in Fig. 16. Example 7 Characterization of M197X Variants As can be seen in Fig. 9, there was a wide range of amylase activity (measured in the soluble substrate assay) expressed by the M197X (A4 form) variants. The amylases were partially purified from the supernatants by precipitation with two volumes of ethanol and resuspension. They were then screened for thermal stability (Fig. 10) by heating at 95°C for 5 minutes in lOmM acetate buffer pH 5.0, in the presence of 5mM calcium chloride; the A4 wild-type retained 28% of its activity after incubation. For M197W and M197P we were unable to recover active protein from the supernatants. Upon sequencing, the M197H variant was found to contain a second mutation, N190K. M197L was examined in a separate experiment and was one of the lowest thermally stable variants. There appears to be a broad correlation between expression of amylase activity and thermal stability. The licheniformiε amylase is restricted in what residues it can accommodate at position 197 in terms of retaining or enhancing thermal stability: cysteine and threonine are preferred for maximal thermal stability under these conditions whereas alanine and isoleucine are of intermediate stability. However, other substitutions at position +197 result in lowered thermal stability which may be useful for other applications. Additionally, different substitutions at +197 may have other beneficial properties, such as altered pH performance profile or altered oxidative stability. For example, the M197C variant was found to inactivate readily by air oxidation but had enhanced thermal stability. Conversely, compared to the M197L variant, both M197T and M197A retained not only high thermal stability (Fig. 10), but also high activity (Table III), while maintaining resistance to inactivation by peroxide at pH 5 to pH 10 (Fig. 7) . Example 8 Stability and Performance in Detergent Formulation The stability of the M197T (A4 form) , M197T and M197A (A4 form) was measured in automatic dish care detergent (ADD) matrices. 2ppm Savinase™ (a protease, commercially available from Novo Industries, of the type commonly used in ADD) were added to two commercially available bleach-containing ADD's: Cascade™ (Procter and Gamble, Ltd.) and Sunlight™ (Unilever) and the time course of inactivation of the amylase variants and Termamyl™ (a thermally stable alpha- amylase available from Novo Nordisk, A/S) followed at 65°C. The concentration of ADD product used in both cases was equivalent to 'pre-soak' conditions: 14gm product per liter of water (7 grams per gallon hardness). As can be seen (Figs. 11a and lib), both forms of the M197T variant were much more stable than Termamyl ,,, and M197A (A4 form) , which were inactivated before the first assay could be performed. This stability benefit was seen in the presence or absence of starch as determined by the following protocol. Amylases were added to 5ml of ADD and Savinase™, prewarmed in a test tube and, after vortexing, activities were assayed as a function of time, using the soluble substrate assay. The "+ starch" tube had spaghetti starch baked onto the sides (140°C, 60 mins.). The results are shown in Figs. 11a and lib. Example 9 Characterization of M15X Variants All M15X variants were propagated in Bacillus suJ tilis and the expression level monitored as shown in Fig. 13. The amylase was isolated and partially purified by a 20-70% ammonium sulfate cut. The specific activity of these variants on the soluble substrate was determined as per Example 3 (Fig. 14). Many of the M15X amylases have specific activities greater than that of SpezymeΦ AA20. A benchtop heat stability assay was performed on the variants by heating the amylase at 90°C for 5 min. in 50 mM acetate buffer pH 5 in the presence of 5 mM CaCl 2 (Fig. 15) . Most of the variants performed as well as SpezymeΦ AA20 in this assay. Those variants that exhibited reasonable stability in this assay (reasonable stability defined as those that retained at least about 60% of SpezymeΦ AA20's heat stability) were tested for specific activity on starch and for liquefaction performance at pH 5.5. The most interesting of those mutants are shown in Fig. 16. M15D, N and T, along with L, outperformed SpezymeΦ AA20 in liquefaction at pH 5.5 and have increased specific activities in both the soluble substrate and starch hydrolysis assays. Generally, we have found that by substituting for the methionine at position 15, we can provide variants with increased low pH- liquefaction performance and/or increased specific activity. Example 10 Tryptophan Sensitivity to Oxidation Chloramine-T (sodium N-chloro-p-toluenesulfonimide) is a selective oxidant, which oxidizes methionine to methionine sulfoxide at neutral or alkaline pH. At acidic pH, chloramine-T will modify both methionine and tryptophan (Schechter, Y. , Burstein, Y. and Patchornik, A. (1975) Biochemistry 14 (20) 4497-4503). Fig. 17 shows the inactivation of B. licheniformiε alpha-amylase with chloramine-T at pH 8.0 (AA20 = 0.65 mg/ml, M197A = 1.7 mg/ml, M197L = 1.7 mg/ml). The data shows that by changing the methionine at position 197 to leucine or alanine, the inactivation of alpha- amylase can be prevented. Conversely, as shown in Fig. 18, at pH 4.0 inactivation of the M197A and M197L proceeds, but require more equivalents of chloramine-T (Fig. 18; AA20 = 0.22 mg/ml, M197A = 4.3 mg/ml, M197L = 0.53 mg/ml; 200 mM NaAcetate at pH 4.0). This suggests that a tryptophan residue is also implicated in the chloramine-T mediated inactivation event. Furthermore, tryptic mapping and subsequent amino acid sequencing indicated that the tryptophan at position 138 was oxidized by chloramine-T (data not shown) . To prove this, site-directed mutants were made at tryptophan 138 as provided below: Preparation of Alpha-A vlase Double Mutants W138 and M197 Certain variants of W138 (F, Y and A) were made as double mutants, with M197T (made as per the disclosure of Example 3). The double mutants were made following the methods described in Examples 1 and 3. Generally,single negative strands of DNA were prepared from an M13MP18 clone of the 1.72kb coding sequence (Pst I-Sst I) of the B. licheniformiε alpha-amylase M197T mutant. Site-directed mutagenesis was done using the primers listed below, essentially by the method of Zoller, M. et al. (1983) except T4 gene 32 protein and T4 polymerase were substituted for klenow. The primers all contained unique sites, as well as the desired mutation, in order to identify those clones with the appropriate mutation. Tryptophan 138 to Phenvlalanine 133 134 135 136 137 138 139 140 141 142 143 CAC CTA ATT AAA GCT C ACA CAT TTT CAT TTT Seq ID No 42 Hind III Tryptophan 138 to Tvrosine 133 134 135 136 137 138 139 140 141 142 143 CAC CTA ATT AAA GCT TAC ACA CAT TTT CAT TTT Seq ID No 43 Hind III Tryptophan 138 to Alanine - This primer also engineers unique sites upstream and downstream of the 138 position. 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 C CGC GTA ATT TCC GGA GAA CAC CTA ATT AAA GCC OCA ACA CAT TTT CAT BspE I 143 144 145 146 147 TTT CCC GGG CGC GGC AG Seq ID No 44 Xma I Mutants were identified by restriction analysis and W138F and W138Y confirmed by DNA sequencing. The W138A sequence revealed a nucleotide deletion between the unique BspE I and Xma I sites, however, the rest of the gene sequenced correctly. The 1.37kb Sstll/Sstl fragment containing both W138X and M197T mutations was moved from M13MP18 into the expression vector pBLapr resulting in pBLapr (W138F, M197T) and pBLapr (W138Y, M197T) . The fragment containing unique BspE I and Xma I sites was cloned into pBLapr (BspE I, Xma I, M197T) since it is useful for cloning cassettes containing other amino acid substitutions at position 138. Single Mutations at Amino Acid Position 138 Following the general methods described in the prior examples, certain single variants of W138 (F, Y, L, H and C) were made. The 1.24kb Asp718-Sstl fragment containing the M197T mutation in plasmid pBLapr (W138X, M197T) of Example 7 was replaced by the wild- type fragment with methionine at 197, resulting in pBLapr (W138F) , pBLapr (W138Y) and pBLapr (BspE I, Xma I) . The mutants W138L, W138H and W138C were made by ligating synthetic cassettes into the pBLapr (BspE I, Xma I) vector using the following primers: Tryptophan 138 to Leucine CC GGA GAA CAC CTA ATT AAA GCC CTA ACA CAT TTT CAT TTT C Seq ID No 45 Tryptophan 138 to Histidine CC GGA GAA CAC CTA ATT AAA GCC CAC ACA CAT TTT CAT TTT C Seq ID No 46 Tryptophan 138 to Cvsteine CC GGA GAA CAC CTA ATT AAA GCC TβC ACA CAT TTT CAT TTT C Seq ID No 47 Reaction of the double mutants M197T/W138F and M197T/W138Y with chloramine-T was compared with wild-type (AA20 = 0.75 mg/ml, M197T/W138F = 0.64 mg/ml, M197T/W138Y = 0.60 mg/ml; 50 mM NaAcetate at pH 5.0) . The results shown in Fig. 19 show that mutagenesis of tryptophan 138 has caused the variant to be more resistant to chloramine-T. Example 11 Preparation of Multiple Mutants Following the methods of Examples 1, 3, 5 and 10, the following multiple mutants were made: M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T and M15T/W138Y/M197T. Certain of these multiple mutants were previously exemplified, for example, W138Y/M197T was made and tested in Example 10. The multiple mutants were identified by restriction analysis. Various multiple mutants within the scope of the present invention were further tested for performance as cleaning products (automatic dish care detergent) additives. These tests are detailed below. Stability Testing A 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg. Certain amylase mutants described above were added to this ADD solution to yield a rate of 0.4 when assayed by the Ceralpha method (Megazyme (Austr.) Pty. Ltd., Parramatta, NSW, Australia) . One set of samples was held at room temperature (21- 23°C) for about 30 min. (non-heated) . A second set of samples was warmed from room temperature to about 65°C after addition of the enzyme (heated) . 30 min. after addition of the enzyme, the activity of the amylase mutants was measured and the activity relative to the activity at the time of addition of the enzyme was calculated (relative activity %) . The results shown in Fig. 20 indicate that the methionine at position +197 of B. licheniformiε alpha-amylase should be modified for stability in a formulation comprising ADD + perborate + TAED. Starch Hydrolysis Assay A 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg and three cooked pieces of elbow macaroni were added. The amylase mutants described above were added to this ADD solution to yield a final concentration of 5 ppm active enzyme. The tubes were incubated at 50°C for about 30 min. and the concentration of reducing sugars released was measured against a glucose standard curve using the dinitrosalicylic acid method. Results are shown in Table VI. Table V The results shown in Table VI show that M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T and M15T/W138Y/M197T performed well compared to no enzyme and wild-type alpha-amylase controls. Oatmeal Stains Dishes were evenly soiled with a cooked, blended oatmeal paste and dried overnight at 37°C. Dishes were loaded in an ASKO Model 770 dishwasher and washed at 45°C on the Quick Wash cycle using 10 g of automatic dishwashing detergent containing 5% perborate, 3% TAED and 11 mg of certain amylase enzyme(s) . The plates were weighed before soiling, after soiling and after washing, and the average % soil removed from all plates was calculated. The data are shown below in Table VII. Table VII The data show that the mutant enzymes provided a benefit greater than that provided by the wild-type. Wild-type amylase provided a 20% greater cleaning benefit in removing oatmeal than did ADD without amylase. Example 12 Dish Care Cleaning Composition 1% (w/w) granules of wild-type and mutant amylases were formulated with a Korex Automatic Dishwasher Detergent to which 5% (w/w) sodium perborate monohydrate and 3% (w/w) TAED were added. Samples of these formulations were placed at room temperature (21-23 β C) or at 38°C and 80% relative humidity for four weeks. Results are shown in Figs. 21 and 22. The data show that the wild-type amylase activity, as measured by the Ceralpha method, decreased with increasing storage time in detergent. At room temperature, the mutant enzymes were completely stable. At 38 C C and 80% relative humidity, all mutants were more stable than the wild-type. The advantage of formulating an automatic dishwashing detergent with these mutant amylases is that these mutants are significantly more stable than the wild-type in the presence of perborate and TAED and they provide a significant performance benefit in removing starchy food stains in the wash. Example 13 Oxidatively Stable Protease/Oxidatively Stable Amylase Stability Studies Enzyme granules containing either: 1) wild-type protease and wild- type amylase; or 2) bleach stable protease (GG36-M222S) made by the methods described in US Re 34606 and bleach stable amylase (AA20- M15T/W138Y/M197T) were dissolved in buffer containing 0.1 M sodium borate pH 10.2 and 0.005% Tween 80 at a concentration of 12.5 mg of each enzyme. To 9 ml of these solutions was added either 1 ml distilled water or 1 ml 30% hydrogen peroxide. After incubation of the solutions at 25 β C for 30 minutes, the protease and amylase activity in each solution was measured and is reported as % of the original activity. The data are shown below in Table VIII. Table VIII Treatment Enzyme \ Activity After 30 Min Water Water Water Water 3% Peroxide 3% Peroxide 3% Peroxide 3% Peroxide The data show that the combination of a bleach-stable amylase mutant and a bleach-stable protease mutant, both with mutations at amino acid residues sensitive to oxidation, provides the combined benefits of protease and amylase in a formulation resistant to inactivation by bleach. The combination of a bleach-stable amylase and a bleach- stable protease retains most of its initial activity after 30 minutes in bleach, while the combination of wild-type enzymes loses over 80% of its initial activity in the same period of time. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Bamett, Christopher Hitchinson, Colin Power, Scott D. (ii) TITLE OF INVENTION: An Improved Cleaning Composition (iii) NUMBER OF SEQUENCES: 6B (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genencor International (B) STREET: 180 Kimball Way (C) CITY: South San Francisco (D) STATE: CA (E) COUNTRY: USA (F) ZIP: 94080 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNE /AGENT INFORMATION: (A) NAME: Horn, Margaret A. (B) REGISTRATION NUMBER: 33,401 (C) REFERENCE/DOCKET NUMBER: GC220-3 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 742-7536 (B) TELEFAX: (415) 742-7217 (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: GATCAAAACA TAAAAAACCG GCCTTGGCCC CGCCGGTTTT TTATTATTTT TGAGCT 56 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:2: TGGGACGCTG GCGCAGTACT TTGAATGGT 29 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:3: TGATGCAGTA CTTTGAATGG TACCTGCCCA ATGA 34 (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4: GATTATTTGT TGTATGCCGA TATCGACTAT GACCAT 36 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs. (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CGGGGAAGGA GGCCTTTACG GTAGCT 26 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCGGCTATGA CTTAAGGAAA TTGC 24 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH.* 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTACGGGGAT GCATACGGGA CGA 23 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTACGGGGAT TACTACGGGA CCAAGGGAGA CTCCC 35 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCGGTGGGGC CAAGCGGGCC TATGTTGGCC GGCAAA 36 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:10: CATCAGCGTC CCATTAAGAT TTGCAGCCTG CGCAGACATG TTGCT 45 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:ll: GATTATTTGG CGTATGCCGA TATCGACTAT GACCAT 36 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAAGTTTC GAATGAAAAC G 21 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTCGGCATAT GCATATAATC ATAGTTGCCG TTTTCATT 38 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CGAATGAAAA CGGCAACTAT GATTATTTGA TCTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:15: CGAATGAAAA CGGCAACTAT GATTATTTGT TCTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CGAATGAAAA CGGCAACTAT GATTATTTGG TTTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGAATGAAAA CGGCAACTAT GATTATTTGA GCTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGAATGAAAA CGGCAACTAT GATTATTTGC CTTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:19: CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CGAATGAAAA CGGCAACTAT GATTATTTGT ACTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs . (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:21: CGAATGAAAA CGGCAACTAT GATTATTTGC ACTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CGAATGAAAA CGGCAACTAT GATTATTTGG GCTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CGAATGAAAA CGGCAACTAT GATTATTTGC AATATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CGAATGAAAA CGGCAACTAT GATTATTTGA ACTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GCAATGAAAA CGGCAACTAT GATTATTTGA AATATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: . (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CGAATGAAAA CGGCAACTAT GATTATTTGG ATTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CGAATGAAAA CGGCAACTAT GATTATTTGG AATATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CGAATGAAAA CGGCAACTAT GATTATTTGT GTATTGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CGAATGAAAA CGGCAACTAT GATTATTTGT GGTATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1968 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:31 AGCTTGAAGA AGTGAAGAAG CAGAGAGGCT ATTGAATAAA GGCGCTTTTC TTTTGGAAGA AAATATAGGG AAAATGGTAC TTATACAACA TCATATGTTT CACATTGAAA GGGGAGGAGA GCTTTACGCC CGATTGCTGA CGCTGTTATT TGCGCTCATC AGCAGCGGCG GCAAATCTTA ATGGGACGCT GATGCAGTAT TGACGGCCAA CATTGGAAGC GTTTGCAAAA CGACTCGGCA TACTGCCGTC TGGATTCCCC CGGCATATAA GGGAACGAGC TGCTTACGAC CTTTATGATT TAGGGGAGTT TCATCAAAAA CGGCACAAAA GGAGAGCTGC AATCTGCGAT CAAAAGTCTT TTACGGGGAT GTGGTCATCA ACCACAAAGG CGGCGCTGAT GGTTGAAGTC GATCCCGCTG ACCGCAACCG CGTAATTTCA CTGGACACAT TTTCATTTTC CGGGGCGCGG CAGCACATAC GTACCATTTT GACGGAACCG ATTGGGACGA GTCCCGAAAG TCAAGGAAAG GCTTGGGATT GGGAAGTTTC CAATGAAAAC GTATGCCGAC ATCGATTATG ACCATCCTGA TGTCGCAGCA TTGGTATGCC AATGAACTGC AATTGGACGG TTTCCGTCTT ATTTTCTTTT TTGCGGGATT GGGTTAATCA TGTCAGGGAA TACGGTAGCT GAATATTGGC AGAATGACTT GGGCGCGCTG AAATTTTAAT CATTCAGTGT TTGACGTGCC GCTTCATTAT ACAGGGAGGC GGCTATGATA TGAGGAAATT GCTGAACGGT GTTGAAATCG GTTACATTTG TCGATAACCA TGATACACAG GACTGTCCAA ACATGGTTTA AGCCGCTTGC TTACGCTTTT ATACCCTCAG GTTTTCTACG GGGATATGTA CGGGACGAAA TCCTGCCTTG AAACACAAAA TTGAACCGAT CTTAAAAGCG AGCACAGCAT GATTATTTCG ACCACCATGA CATTGTCGGC CTCGGTTGCA AATTCAGGTT TGGCGGCATT AATAACAGAC AATGTATGTC GGCCGGCAAA ACGCCGGTGA GACATGGCAT GGAGCCGGTT GTCATCAATT CGGAAGGCTG GGGAGAGTTT TTCAATTTAT GTTCAAAGAT AGAAGAGCAG AGAGGACGGA TTTTTATTTT GCCCGTCTTA TAAATTTCTT TGATTACATT GTGTCATCAG CCCTCACGAA GGACTTGCTG ACAGTTTGAA TCGCATAGGT AAGGCGGGGA 1860 TGAAATGGCA ACGTTATCTG ATGTAGCAAA GAAAGCAAAT GTGTCGAAAA TGACGGTATC 1920 GCGGGTGATC AATCATCCTG AGACTGTGAC GGATGAATTG AAAAAGCT 1968 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 483 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Ala Asn Leu Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gin Ser Ala He Lys Ser Leu His Ser Arg Asp He Asn 85 90 95 Val Tyr Gly Asp Val Val He Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 He Ser Gly Glu His Leu He Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg He Tyr Lys 165 170 175 Phe Gin Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp He Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu He Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gin 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His He Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gin Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285 His Tyr Gin Phe His Ala Ala Ser Thr Gin Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gin Pro Gly Gin Ser Leu Glu 325 330 335 Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe He Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gin Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gin Arg Glu He Pro Ala Leu Lys His Lys He 370 375 380 Glu Pro He Leu Lys Ala Arg Lys Gin Tyr Ala Tyr Gly Ala Gin His 385 390 395 400 Asp Tyr Phe Asp His His Asp He Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu He Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gin Asn Ala Gly Glu Thr 435 440 445 Trp His Asp He Thr Gly Asn Arg Ser Glu Pro Val Val He Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser He Tyr 465 470 475 480 Val Gin Arg (2) INFORMATION FOR SEQ ID NO.-33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 511 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Met Lys Gin Gin Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe 1 5 10 15 Ala Leu He Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala λsn Leu 20 25 30 Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro λsn Asp Gly 35 40 45 His Trp Lys Arg Leu Gin Asn λsp Ser λla Tyr Leu λla Glu His Gly 50 55 60 He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly Thr Ser Gin Ala 65 70 75 80 Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe His 85 90 95 Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu Gin 100 105 110 Ser Ala He Lys Ser Leu 115 Val Val He λsn His Lys 130 Ala Val Glu Val Asp Pro 145 150 His Leu He Lys Ala Trp 165 Thr Tyr Ser λsp Phe Lys 180 Trp λsp Glu Ser λrg Lys 195 λla Trp λsp Trp Glu Val 210 Met Tyr λla λsp He λsp 225 230 Lys Arg Trp Gly Thr Trp 245 Arg Leu λsp λla Val Lys 260 Val λsn His Val λrg Glu 275 Glu Tyr Trp Gin λsn λsp 290 Thr λsn Phe λsn His Ser 305 310 His Ala Ala Ser Thr Gin 325 Asn Gly Thr Val Val Ser 340 Asp Asn His Asp Thr Gin 355 Thr Trp Phe Lys Pro Leu 370 Gly Tyr Pro Gin Val Phe 385 390 Ser Gin Arg Glu He Pro 405 Lys λla λrg Lys Gin Tyr 420 His His λsp He Val Gly 435 λsn Ser Gly Leu λla λla 450 λrg Met Tyr Val Gly λrg 465 470 Thr Gly λsn λrg Ser Glu 485 Glu Phe His Val λsn Gly 500 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARλCTERISTICS: (A) LENGTH: 520 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Met Arg Gly Arg Gly Asn Met He Gin Lys Arg Lys Arg Thr Val Ser 1 5 10 15 Phe λrg Leu Val Leu Met Cys Thr Leu Leu Phe Val Ser Leu Pro He 20 25 30 Thr Lys Thr Ser λla Val λsn Gly Thr Leu Met Gin Tyr Phe Glu Trp 35 40 45 Tyr Thr Pro λsn λsp Gly Gin His Trp Lys λrg Leu Gin λsn λsp λla 50 55 60 Glu His Leu Ser λsp He Gly He Thr λla Val Trp He Pro Pro λla 65 70 75 80 Tyr Lys Gly Leu Ser Gin Ser λsp λsn Gly Tyr Gly Pro Tyr λsp Leu 85 90 95 Tyr λsp Leu Gly Glu Phe Gin Gin Lys Gly Thr Val λrg Thr Lys Tyr 100 105 110 Gly Thr Lys Ser Glu Leu Gin λsp λla He Gly Ser Leu His Ser λrg 115 120 125 λsn Val Gin Val Tyr Gly λsp Val Val Leu λsn His Lys λla Gly λla 130 135 140 λsp λla Thr Glu λsp Val Thr λla Val Glu Val λsn Pro λla λsn λrg 145 150 155 160 λsn Gin Glu Thr Ser Glu Glu Tyr Gin He Lys λla Trp Thr λsp Phe 165 170 175 λrg Phe Pro Gly λrg Gly λsn Thr Tyr Ser λsp Phe Lys Trp His Trp 180 185 190 Tyr His Phe λsp Gly λla λsp Trp λsp Glu Ser λrg Lys He Ser λrg 195 200 205 He Phe Lys Phe λrg Gly Glu Gly Lys λla Trp λsp Trp Glu Val Ser 210 215 220 Ser Glu λsn Gly λsn Tyr λsp Tyr Leu Met Tyr λla λsp Val λsp Tyr 225 230 235 240 λsp His Pro λsp Val Val λla Glu Thr Lys Lys- Trp Gly He Trp Tyr 245 250 255 λla λεn Glu Leu Ser Leu λsp Gly Phe λrg He λsp λla λla Lys His 260 265 270 He Lys Phe Ser Phe Leu λrg λsp Trp Val Gin λla Val λrg G n λla 275 280 285 Thr Gly Lys Glu Met Phe Thr Val λla Glu Tyr Trp Gin λsn λεn λla 290 295 300 Gly Lys Leu Glu λsn Tyr Leu λsn Lys Thr Ser Phe λsn Gin Ser Val 305 310 315 320 Phe λsp Val Pro Leu His Phe λsn Leu Gin λla λla Ser Ser Gin Gly 325 330 335 Gly Gly Tyr λsp Met λrg λrg Leu Leu λsp Gly Thr Val Val Sex λrg 340 345 350 His Pro Glu Lys λla Val Thr Phe Val Glu λsn His λsp Thr Gin Pro 355 360 365 Gly Gin Ser Leu Glu Ser Thr Val Gin Thr Trp Phe Lys Pro Leu λla 370 375 380 Tyr λla Phe He Leu Thr λrg Glu Ser Gly Tyr Pro Gin Val Phe Tyr 385 390 395 400 Gly λsp Met Tyr Gly Thr Lys Gly Thr Ser Pro Lys Glu He Pro Ser 405 410 415 Leu Lys λsp λsn He Glu Pr He Leu Lys λla λrg Lys Glu Tyr λla 420 425 430 Tyr Gly Pro Gin His λsp Tyr He λsp His Pro λsp Val He Gly Trp 435 440 445 Thr λrg Glu Gly λsp Ser Ser Ala Ala Lys Ser Gly Leu λla λla Leu 450 455 460 He Thr λsp Gly Pro Gly Gly Ser Lys λrg Met Tyr λla Gly Leu Lys 465 470 475 480 Asn Ala Gly Glu Thr Trp Tyr Asp He Thr Gly Asn Arg Ser Asp Thr 485 490 495 Val Lys He Gly Ser λsp Gly Trp Gly Glu Phe His Val λsn λsp Gly 500 505 510 Sex Val Ser He Tyr Val Gin Lys 515 520 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 548 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: Val Leu Thr Phe His Arg He He Arg Lys Gly Trp Met Phe Leu Leu 1 5 10 15 λla Phe Leu Leu Thr λla Ser Leu Phe Cys Pro Thr Gly λrg His λla 20 25 30 Lys λla λla λla Pro Phe λsn Gly Thr Met Met Gin Tyr Phe Glu Trp 35 40 45 Tyr Leu Pro λsp λsp Gly Thr Leu Trp Thr Lys Val λla λsn Glu λla 50 55 60 λsn λsn Leu Ser Ser Leu Gly He Thr λla Leu Ser Leu Pro Pro λla 65 70 75 80 Tyr Lys Gly Thr Ser λrg Ser λsp Val Gly Tyr Gly Val Tyr λsp Leu 85 90 95 Tyr λsp Leu Gly Glu Phe λsn Gin Lys Gly Thr Val λrg Thr Lys Tyr 100 105 110 Gly Thr Lys λla Gin Tyr Leu Gin λla He Gin λla λla His λla λla 115 120 125 Gly Met Gin Val Tyr λla λsp Val Val Phe λsp His Lys Gly Gly λla 130 135 140 λsp Gly Thr Glu Trp Val λsp λla Val Glu Val λsn Pro Ser λsp λrg 145 150 155 160 λsn Gin Glu He Ser Gly Thr Tyr Gin He Gin λla Trp Thr Lys Phe 165 170 175 λsp Phe Pro Gly λrg Gly λsn Thr Tyr Ser Ser Phe Lys Trp λrg Trp 180 185 190 Tyr His Phe λsp Gly Val λsp Trp λsp Glu Ser λrg Lys Leu Ser λrg 195 200 205 He Tyr Lys Phe λrg Gly He Gly Lys λla Trp λsp Trp Glu Val λsp 210 215 220 Thr Glu λsn Gly λsn Tyr λεp Tyr Leu Met Tyr λla λsp Leu λsp Met 225 230 235 240 Asp His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr 245 250 255 Val Asn Thr Thr Asn He Asp Gly Phe Arg Leu Asp Gly Leu Lys His 260 265 270 He Lys Phe Ser Phe Phe Pro λsp Trp Leu Ser Tyr Val λrg Ser Gin 275 280 285 Thr Gly Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr λsp He 290 295 300 λsn Lys Leu His λsn Tyr He Thr Lys Thr λεn Gly Thr Met Ser Leu 305 310 315 320 Phe λsp Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly 325 330 335 Gly Ala Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys λsp 340 345 350 Gin Pro Thr Leu Ala Val Thr Phe Val Asp λsn His λsp Thr λsn Pro 355 360 365 λla Lys λrg Cys Ser His Gly λrg Pro Trp Phe Lys Pro Leu λla. Tyr 370 375 380 λla Phe He Leu Thr λrg Gin Glu Gly Tyr Pro Cys Val Phe Tyr Gly 385 390 395 400 λsp Tyr Tyr Gly He Pro Gin Tyr λsn He Pro Ser Leu Lys Ser Lys 405 410 415 He λsp Pro Leu Leu He λla λrg λrg λsp Tyr λla Tyr Gly Thr Gin 420 425 430 His λsp Tyr Leu λsp His Ser λsp He He Gly Trp Thr λrg Glu Gly 435 440 445 Val Thr Glu Lys Pro Gly Ser Gly Leu λla λla Leu He Thr λsp Gly 450 455 460 λla Gly λrg Ser Lys Trp Met Tyr Val Gly Lys Gin His λla Gly Lys 465 470 475 460 Val Phe Tyr λsp Leu Thr Gly λsn λrg Ser λsp Thr Val Thr He λsn 485 490 495 Ser λsp Gly Trp Gly Glu Phe Lys Val λsn Gly Gly Ser Val Ser Val 500 505 510 Trp Val Pro λrg Lys Thr Thr Val Ser Thr He λla λrg Pro He Thr 515 520 525 Thr λrg Pro Trp Thr Gly Glu Phe Val λrg Trp His Glu Pro λrg Leu 530 535 540 Val λla Trp Pro 545 (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 463 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Ala Asn Leu Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gin Ser λla He Lys Ser Leu His Ser λrg λsp He λsn 85 90 95 Val Tyr Gly λsp Val Val He λsn His Lys Gly Gly λla λsp λla Thr 100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg λsn λrg Val 115 120 125 He Ser Gly Glu His Leu He Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser λsp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 λsp Gly Thr λsp Trp Asp Glu Ser Arg Lys Leu Asn Arg He Tyr Lys 165 170 175 Phe Gin Gly Lys Ala Trp λsp Trp Glu Val Ser λsn Glu λsn Gly λsn 180 185 190 Tyr λsp Tyr Leu Thr Tyr λla λsp He λsp Tyr λsp His Pro λsp Val 195 200 205 λla λla Glu He Lys λrg Trp Gly Thr Trp Tyr λla λsn Glu Leu Gin 210 215 220 Leu λsp Gly Phe λrg Leu λsp λla Val Lys His He Lys Phe Ser Phe 225 230 235 240 Leu.λrg λsp Trp Val λsn His Val λrg Glu Lys Thr Gly Lyε Glu Met 245 250 255 Phe Thr Val λla Glu Tyr Trp Gin λsn λsp Leu Gly λla Leu Glu λsn 260 265 270 Tyr Leu λsn Lys Thr λsn Phe λsn His Ser Val Phe λsp Val Pro Leu 275 280 285 His Tyr Gin Phe His λla λla Ser Thr Gin Gly Gly Gly Tyr λsp Met 290 295 300 λrg Lys Leu Leu λsn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val λsp λsn His λsp Thr Gin Pro Gly Gin Ser Leu Glu 325 330 335 Ser Thr Val Gin Thr Trp Phe Lys Pro Leu λla Tyr λla Phe He Leu 340 345 350 Thr λrg Glu Ser Gly Tyr Pro Gin Val Phe Tyr Gly λsp Met Tyr Gly 355 360 365 Thr Lys Gly λsp Ser Gin λrg Glu He Pro λla Leu Lys His Lys He 370 375 380 Glu Pro He Leu Lys λla λrg Lys Gin Tyr λla Tyr Gly λla Gin His 385 390 395 400 Asp Tyr Phe Asp His His Asp He Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu He Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gin Asn λla Gly Glu Thr 435 440 445 Trp His λsp He Thr Gly λsn λrg Ser Glu Pro Val Val He λsn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val λsn Gly Gly Ser Val Ser He Tyr 465 470 475 480 Val Gin λrg (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHλRACTERISTICS: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: λla λla λla λla λla λsn Leu λsn Gly Thr Leu Met Gin Tyr Phe Glu 1 5 10 15 Trp Tyr Met Pro λsn λsp Gly Gin His Trp Lys λrg Leu Gin λsn λsp 20 25 30 Ser λla Tyr Leu λla Glu His Gly He Thr λla Val Trp He Pro Pro 35 40 45 λla Tyr Lys Gly Thr Ser Gin λla λsp Val Gly Tyr Gly λla Tyr λsp 50 55 60 Leu Tyr λsp Leu Gly Glu Phe His Gin Lys Gly Thr Val λrg Thr Lys 65 - 70 75 80 Tyr Gly Thr Lys Gly Glu Leu Gin Ser λla He Lys Ser Leu His Ser 85 90 95 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 32 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Met Lys Gin Gin Lys λrg Leu Thr λla λrg Leu Leu Thr Leu Leu Phe 1 5 10 15 λla Leu He Phe Leu Leu Pro His Ser λla λla λla λla λla λsn Leu 20 25 30 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Met Arg ' Ser Lys Thr Leu Trp He Ser Leu Leu Phe λla Leu Thr Leu 1 5 10 15 He Phe Thr Met λla Phe Ser λsn Met Ser Ala Gin Ala Ala Gly Lys 20 25 30 Ser (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Met Arg Ser Lys Thr Leu Trp He Ser Leu Leu Phe λla Leu Thr Leu 1 5 10 15 He Phe Thr Met λla Phe Ser λsn Met Ser λla Gin λla λla λla λla 20 25 30 λla λla λsn 35 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:41: Met λrg Ser Lys Thr Leu Trp He Ser Leu Leu Phe λla Leu Thr Leu 1 5 10 15 He Phe Thr Met λla Phe Ser λsn Met Ser λla Gin λla λla λsn Leu 20 25 30 (2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRλNDEDNESS: single (D) TOPOLOGY: linear ≠ (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CACCTAATTA AλGCTTTCAC λCλTTTTCAT TTT 33 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO.-43: CACCTAATTA AAGCTTACAC ACATTTTCAT TTT 33 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CCGCGTAλTT TCCGGAGAλC λCCTλATTλA λGCCGCλλCλ CλTTTTCλTT TTCCCGGGCG 60 CGGCAG 66 (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRλNDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: CCGGAGAACA CCTAATTAλA GCCCTAACAC ATTTTCλTTT TC 42 (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CCGGAGAACA CCTAATTAAA GCCCACλCλC λTTTTCλTTT TC 42 (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARλCTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CCGGAGAACA CCTAATTAAA GCCTGCACAC ATTTTCλTTT TC 42 (2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHλRACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GATGCAGTAT TTCGAACTGG TATA 24 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRλNDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TGCCCAATGA TGGCCAACAT TGGλλG 26 (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs " (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: CGAATGGTAT GCTCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CGAATGGTAT CGCCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CGAATGGTAT AATCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CGAATGGTAT GATCCCλλTG λCGG 24 (2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRλNDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CGAATGGTAT CACCCCAATG ACGG 24 (2) INFORMλTION FOR SEQ ID NO:55: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: CGAATGGTλT AAACCCAλTG ACGG 24 (2) INFORMATION FOR SEQ ID NO:56: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGAATGGTAT CCGCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CGAATGGTAT TCTCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CGλλTGGTλC λCTCCCλλTG λCGG 24 (2) INFORMλTION FOR SEQ ID NO:59: (i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRλNDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CGAATGGTAT GTTCCCAλTG λCGG 24 (2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARλCTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: CGAATGGTAT TGTCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: CGAATGGTAT CAACCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: CGAATGGTAT GAλCCCλλTG ACGG 24 (2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARλCTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CGAATGGTAT GGTCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: CGAATGGTAT ATTCCCλλTG λCGG 24 (2) INFORMATION FOR SEQ ID NO:65: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: CGAATGGTAT TTTCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CGAATGGTAC TGGCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CGAλTGGTλT TλTCCCλλTG λCGG 24 (2) INFORMATION FOR SEQ ID NO:68: (i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 base pairs (B) TYPE: nucleic- acid (C) STRλNDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ • ID NO:68: CCGTCATTGG GACTACGTAC CATT 24

Claims: WHAT IS CXΛXMED XS : 1. An improved bleach-containing cleaning composition, the improvement comprising adding to the bleach-containing composition a mutant alpha-amylase that is the expression product of a mutated DNA sequence encoding an alpha-amylase, the mutated DNA sequence being derived from a precursor alpha-amylase by the substitution of a methionine at a position equivalent to M+197 in B. licheniformiε alpha-amylase and the substitution of one or more methionine or tryptophan at a position equivalent to M+15 or W+138 in B. licheniformiε alpha-amylase. 2. An improved cleaning composition of Claim 1 wherein the cleaning composition is a dish care cleaning composition. 3. An improved cleaning composition of Claim 1 wherein the mutant alpha-amylase is selected from the group consisting of M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T and M15T/W138Y/M197T. 4. An improved cleaning composition of Claim 1 further comprising a mutant protease that is the expression product of a mutated DNA sequence encoding a protease, the mutated DNA sequence being derived from a precursor protease by the substitution of a methionine at a position equivalent to M+222 in Bacilluε amyloliquefacienε protease. 5. An improved cleaning composition of Claim 4 wherein the mutant protease comprises a substitution selected from the group of amino acids consisting of alanine, cysteine and serine. 6. An improved cleaning composition of Claim 4 comprising an alpha-amylase mutant selected from the group consisting of M15T/M197T, M15S/M197T, W138Y/M197T, M15S/W138Y/M197T and M15T/W138Y/M197T, and a protease mutant selected from the group consisting of M222C, M222S and M222A. 7. An improved cleaning composition of Claim 6 which is a granular composition.

Inventors: BARNETT CHRISTOPHER C, MITCHINSON COLIN, POWER SCOTT D, BARNETT, CHRISTOPHER, C., MITCHINSON, COLIN, POWER, SCOTT, D.
Applicants: GENENCOR INT, GENENCOR INTERNATIONAL, INC.
Application year: 1995
Main classification: C12N9
IPC codes: C07K14, C11D3, C11D7, C12N9, C12N15, C12R1