AU718509B2 - An improved laundry detergent composition comprising amylase - Google Patents

Description

WO 96/30481 PCT1US96/04029 1 AN IMPROVED LAUNDRY DETERGENT COMPOSITION COMPRISING AMYLASE Field of the Invention The present invention relates to laundry detergents containing novel alpha-amylase mutants having an amino acid sequence not found in nature, such alpha-amylase mutants having an amino acid sequence wherein one or more amino acid residue(s) of a precursor alphaamylase, specifically any oxidizable amino acid, have been substituted with a different amino acid. The mutant enzymes of the laundry detergents 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.

Background of the Invention Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolase, EC3.2.1.1) hydrolyze internal alpha-1,4-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 Aspergillus, with most commercial amylases being produced from bacterial sources such as B. licheniformis, 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 2 al. (1990) J. Bichem. 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 10:157 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 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 SUBSTITUTE SHEET (RULE 26) 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 acid 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.

Summary of the Invention The present invention provides an improved laundry detergent composition, the improvement comprising adding to the laundry detergent 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 alphaamylase gene by the substitution of a methionine at a position equivalent to M+197 in B.licheniformis alpha-amylase and the substitution of one or more methionine or tryptophan at a position equivalent to M+15 or W+138 in B. licheniformis alphaamylase, wherein said detergent has a pH of about 6 to 8.

The present invention relates to novel laundry detergent compositions comprising 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 results 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 substitution of one or more tryptophan residue alone IC W:\llona\Sharon\sp153226.doc or in combination with the substitution of one or more methionine residue in the precursor alpha-amylase. Such mutant alpha-amylases, in general, an obtained by in vitro 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 ortyrosine residues in such sequence, most preferably the residue which is IC W:lona\Sharon\sp153226.doc WO 96/30481 PCT/US96/04029 4 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 cysteine may be substituted for methionine).

Preferred laundry detergents comprise mutants comprising the substitution of a methionine residue equivalent to any of the methionine residues found in B. licheniformis alpha-amylase +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 isoleucine threonine or cysteine The preferred substitute amino acids at position are leucine threonine asparagine aspartate serine valine and isoleucine although other substitute amino acids not specified above may be useful. Two specifically preferred mutants of the present invention are M197T and Another embodiment of this invention relates to laundry detergents comprising mutants comprising the substitution of a tryptophan SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 5 residue equivalent to any of the tryptophan residues found in B.

licheniformis alpha-amylase (see Fig. 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 included in the laundry detergents 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 specifically, 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. Accordingly, a preferred embodiment of the present invention comprises a laundry detergent comprising the mutant alpha-amylases of the invention and further comprising a bleach or peracid compound. A particularly preferred embodiment of the invention is a laundry detergent comprising the mutant alphaamylases according to the invention which has a pH above about and more preferably of between about 10 and about 12. Also preferred is a granular laundry detergent having a pH of between about 10 and about 12 and further containing a bleach or peracid compound.

Mutant enzymes according to the invention are also surprisingly characterized by having superior activity in the neutral pH ranges when compared to wild type or non-inventive amylases. Accordingly, another particularly preferred embodiment comprises a laundry detergent comprising the mutant alpha-amylases of the invention and having a pH of between about 5.0 and about 10.0, more preferably between 6.0 and about 10.0. A most preferred embodiment is a liquid laundry detergent having a pH between about 6.0 and about 10.0.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 6 Similarly, reduced oxidative stability may be useful 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 purpose and formulation of the laundry detergent comprising the mutant alpha-amylase.

The preferred laundry detergents of the invention comprise alphaamylase mutants derived from a Bacillus strain such as B.

licheniformis, B. amyloliquefaciens, and B. stearothermophilus, and most preferably from Bacillus licheniformis.

In another aspect of the present invention there is provided a laundry detergent comprising 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 Nterminus 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 alphaamylase containing one or more additional mutations such as, for example, mutation (substitution, replacement or deletion) of one or more oxidizable amino acid(s).

A composition embodiment of the present invention comprises laundry detergent compositions, liquid, gel or granular, comprising the alpha-amylase mutants described herein. Preferred are detergent compositions comprising a +197 position mutant either alone or in combination with other enzymes such as endoglycosidases, SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 7 cellulases, proteases, lipases or other amylase enzymes.

Particularly preferred are laundry detergent compositions comprising a M15X/W138X/M197X mutant, and most preferably a M15T/W138Y/M197T mutant. Additionally, it is contemplated that the compositions of the present invention may include an alpha-amylase mutant having more than one site-specific mutation.

According to a process embodiment of the present invention, the laundry detergent composition of the present invention is used in a method to clean soiled laundry.

Brief Description of the Drawings Fig. la-lc 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. 3a-3b 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. amyloliquefaciens 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. Biochem. 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 alphaamylase from B. licheniformis NCIB8061, Seq ID No 37. Numbering is from the N-terminus, starting with the four additional alanines.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 8 Fig. 5 shows plasmid pA4BL wherein BLAA refers to B. licheniformis alpha-amylase gene, PstI to SstI; AmpR refers to the ampicillinresistant gene from pBR322; and CAT refers to the Chloramphenicolresistant gene from pC194.

Fig. 6 shows the signal sequence-mature protein junctions for B.

licheniformis (Seq ID No 38), B. subtilis (Seq ID No 39), B.

licheniformis in pA4BL (Seq ID No 40) and B. licheniformis in pBLapr (Seq ID No 41).

Fig. 7a shows inactivation of certain alpha-amylases and M197L (A4 form) with 0.88M H 2 0 2 at pH 5.0, 250C.

Fig. 7b shows inactivation of certain alpha-amylases M197T) with 0.88M H 2 0 2 at pH 10.0, Fig. 7c shows inactivation of certain alpha-amylases M15L) with 0.88M H 2 0 2 at pH 5.0, 250C.

(Spezyme® (Spezyme® (Spezyme® 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, CaCl 2 at 95 0 C for 5 mins.

Figs. lla and llb show inactivation of certain amylases in automatic dish care detergents. Fig. lla shows the stability of certain amylases in CascadeTM (a commercially available dish care product) at 650C in the presence or absence of starch. Fig. llb shows the stability of certain amylases in Sunlight TM (a commercially available dish care product) at 650C in the presence or absence of starch.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 9 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 90C, pH 5.0, CaCl 2 5 mins.

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. licheniformis wild-type.

Fig. 17 shows the inactivation of B. licheniformis alpha-amylase at 0.65 mg/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. licheniformis alpha-amylase 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. licheniformis alpha-amylase 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 alphaamylase multiple mutants incorporated in automatic dish detergent (ADD) formulations at temperatures from room temperature increased to 65 0

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.

SUBSTITUTE SHEET (RULE 26) Fig. 22 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at 38 0 C (100 0 F) with relative humidity over 0-30 days.

Fig. 23 shows the pH activity profile of certain amylases on a Phadebas substrate at 25 0 C at neutral and alkaline pH.

Fig. 24 shows the stability of certain amylases to peracetic acid over time at pH 9.3 and 52*C.

Fig. 25 shows the relative cleaning ability of amylase according to the invention compared to Termamyl amylase in liquid laundry detergent at 40*C in terms of reflectance (delta from control) vs. ppm amylase added.

Fig. 26 shows the relative cleaning-ability of amylase according to the invention compared to Termamyl amylase in liquid laundry detergent at 55*C in terms of reflectance (delta from control) vs. ppm amylase added.

Fig. 27 shows the wash performance of amylase according to the invention in commercially available detergent in terms of reflectance (delta from control) vs. ppm amylase added.

Detailed Description of the Invention 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 patent 5,322,778 and W093/09244 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 peracidcontaining detergents, may result in partial or complete inactivation of the amylase. Therefore, the present invention focuses on altering the oxidative sensitivity of amylases which are added to laundry detergents. The mutant enzymes in the laundry detergents of the present invention may also have an altered pH -11 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. licheniformis or B. stearothermophilus, including the A4 form of alpha-amylase derived from B. licheniformis as described herein, as well as fungal alpha-amylases such as those derived from Aspergillus 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 15 reference.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", are not intended to exclude other additives or components or integers or steps.

Homologies have been found between almost all endo-amylases S 20 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 Bacillus 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 Bacillus endo-amylases (Feng, D.F. and Doolittle, R.F. (1987) J. Molec. Evol. 35:351- 360). The relative sequence homology between B. stearothermophilus and B.

licheniformis amylase is about 66%, as determined by Holm, L. et al. (1990) Protein Engineering 3 pp. 181-191. The sequence homology between B.

licheniformis and B. amyloliquefaciens amylases is about 81%, as per Holm, L. et al., supra. While sequence homology is important, it is generally recognized that structural homology is also important in comparing WO 96/30481 PCT/US96/04029 12 amylases or other enzymes. For example, structural homology between fungal amylases and bacterial (Bacillus) 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 se. 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 Bacillus licheniformis alpha-amylase are identified herein for substitution or deletion, as are all methionine, histidine, tryptophan, cysteine and tyrosine positions. The amino acid position number +197) refers to the number assigned to the mature Bacillus licheniformis alpha-amylase sequence presented in Fig. 2. The invention, however, is not limited to the mutation of this particular mature alpha-amylase licheniformis) but extends to precursor alpha-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in B. licheniformis alpha-amylase. A residue (amino acid) of a precursor alpha-amylase is equivalent to a residue of B.

licheniformis alpha-amylase if it is either homologous corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. licheniformis alpha-amylase having the same or similar SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 13 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. licheniformis 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 Aspergillus 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 Bacillus 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. stearothermophilus enzyme has been modeled on that of Takaamylase 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 licheniformis 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 mRNA ribosome-binding sites, and sequences which control termination of transcription and translation. A preferred promoter is the B. subtilis aprE promoter. The vector may be a plasmid, a phage particle, or simply SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 14 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 amylases produced by 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 Bacillus strain. Preferably an alpha-amylase negative Bacillus strain (genes deleted) and/or an alpha-amylase and protease deleted Bacillus strain such as Bacillus subtilis strain BG2473 (AamyE,Aapr,Anpr) 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 laundry detergent cleaning compositions, and especially those cleaning compositions containing known oxidants, such as bleach or peracid compounds. Alpha-amylase mutants of the invention can be formulated into known powdered, liquid or gel detergents having pH between about 4.5 to about 12.0, preferably SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 15 between about 5.0 and about 10.0 and most preferably between about and about 10.0. An additional preferred embodiment comprises laundry detergents having a pH of between about 10.0 and about 12.0, wherein bleach is present in the composition. Suitable granular amylase containing compositions 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 detergent products, and particularly laundry detergent 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 proteases, lipases, cellulases, etc.), and preferably oxidatively stable proteases.

Suitable oxidatively stable proteases include genetically engineered proteases such as those described in US Re 34,606, 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. amyloliquefaciens, are described in US Re 34606. Suitable methods for determining SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 16 "equivalent" positions in other subtilisins are provided in Re 34606, EP 257,446 and USSN 212,291, which are incorporated herein by reference.

Abbreviations used herein, particularly three letter or one letter notations for amino acids are described in Dale, Molecular Genetics of Bacteria, John Wiley Sons, (1989) Appendix B.

Experimental Example 1 Substitutions for the Methionine Residues in B. licheniformis Alpha-Amylase The alpha-amylase gene (Fig. 1) was cloned from B. licheniformis NCIB8061 obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (Gray, G. et al. (1986) J.

Bacteriology 166:635-643). The 1.72kb PstI-SstI 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 BclI and SstI sites using a synthetic oligonucleotide cassette of the form: BclI SstI GATCAAAACATAAAAAACCGGCCTTGGCCCCGCCGGTTTTTTATTATTTTTGAGCT 3' 3' TTTTGTATTTTTTGGCCGGAACCGGGGCGGCCAAAAATAATAAAAAC Seq ID No 1 designed to contain the B. amyloliquefaciens 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PTU9/42 PCT/US96/04029 17 for each of the seven methionines found in B. lichenifforinis alphaamylase. Each mutagenic oligonucleotide also introduced a restriction endonuclease site to use as a screen for the linked mutation.

TABLE I Mutagenic Oligonucleotides for the Substitution of the Methionine Residues in B. licheniformis Alpha-Axnylase

MBA

GGG ACG CTG GCG CAG TAC TTT GAA TGG T-31 ScaI+ ATG CAG TAC TTT GAA TGG TAC CTG CCC AAT GA-31 ScaI+ KpnI+ M197L TAT TTG TTG TAT GCC GAT ATC GAC TAT GAC CAT-31 EcoRV+ M256A GGG AAG GAG GCC TTT ACG GTA GCT-3v StuI+ Seq ID No 2 Seq ID No 3 Seq ID No 4 Seq ID No M304L GGC TAT GAC TTA AGG AAA TTG C-3' Af III M366A TAC GGG GAT GCA TAC GGG ACG A-3' Ns iI+ Seq ID No 6 Seq ID No 7 M366Y TAC GGG GAT TAC TAC GGG ACC AAG GGA GAC TCC C-3' StyI+ M438A GGT GGG GCC AAG CGG GCC TAT GTT GGC CGG CAA A-3' Sfii+ Seq ID No 8 Seq ID No 9 Bold letter indicate base changes introduced by oligonucleotide.

Codon changes indicated in the form MBA, where methionine at position +8 has been changed to alanine SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 -18 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 PstI-SstI fragments for each subcloned into an E. coli vector, plasmid pA4BL.

Plasmid pA4BL Following the methods described in US application 860,468 (Power et which is incorporated herein by reference, a silent PstI site was introduced at codon +1 (the first amino-acid following the signal cleavage site) of the aprE gene from pS168-1 (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 pJH101 plasmid (Ferrari, F.A. et al. (1983) J. Bacter. 154:1513-1515) as a HindIII-PstI fragment and subcloned into the pUC18-derived plasmid JM102 (Ferrari, E. and Hoch, J.A. (1989) Bacillus, ed. C.R.

Harwood, Plenum Pub., pp. 57-72). Addition of the PstI-SstI fragment from B. licheniformis alpha-amylase gave pA4BL (Fig. having the resulting aprE signal peptide-amylase junction as shown in Fig. 6.

Transformation Into B. subtilis pA4BL is a plasmid able to replicate in E. coli and integrate into the B. subtilis chromosome. Plasmids containing different variants were transformed into B. subtilis (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 Bacillus subtilis strain BG2473 was a derivative of 1168 which had been deleted for amylase (AamyE) and two proteases (Aapr, Anpr) (Stahl, M.L. and Ferrari, J. Bacter. 158:411-418 and US Patent 5,264,366, incorporated herein by reference). After transformation the sacU32(Hy) (Henner, D.J. et al. (1988) J. Bacter. 170:296-300) SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 -19 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.

subtilis 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 experiments the correctly processed forms of the licheniformis amylase and the variant M197T were made from a very similar construction (see Fig.

Specifically, the 5' end of the A4 construction was subcloned on an EcoRI-SstII fragment, from pA4BL (Fig. 5) into M13BM20 (Boehringer Mannheim) in order to obtain a coding-strand template for the mutagenic oligonucleotide below: CAG CGT CCC ATT AAG ATT TGC AGC CTG CGC AGA CAT GTT GCT-3' Seq ID No This primer eliminated the codons for the extra four N-terminal alanines, correct forms being screened for by the absence of the PstI site. Subcloning the EcoRI-SstII fragment back into the pA4BL vector (Fig. 5) gave plasmid pBLapr. The M197T substitution could then be moved, on a SstII-SstI fragment, out of pA4BL (M197T) into the complementary pBLapr vector to give plasmid pBLapr (M197T). Nterminal analysis of the amylase expressed from pBLapr in B.

subtilis showed it to be processed with the same N-terminus found in B. licheniformis alpha-amylase.

Example 2 Oxidative Sensitivity of Methionine Variants B. licheniformis alpha-amylase, such as Spezyme® AA20 (commercially available from Genencor International, Inc.), is inactivated rapidly in the presence of hydrogen peroxide (Fig. Various methionine variants were expressed in shake-flask cultures of B.

subtilis and the crude supernatants purified by ammonium sulphate SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 20 cuts. The amylase was precipitated from a 20% saturated ammonium sulphate supernatant by raising the ammonium sulphate to saturated, and then resuspended. The variants were then exposed to 0.88M hydrogen peroxide at pH 5.0, at 25 0 C. Variants at six of the methionine positions in B. licheniformis alpha-amylase were still subject to oxidation by peroxide while the substitution at position +197 (M197L) showed resistance to peroxide oxidation. (See Fig.

However, subsequent analysis described in further detail below showed that while a variant may be susceptible to oxidation at pH 25 0 C, it may exhibit altered/enhanced properties under different conditions liquefaction).

Example 3 Construction of All Possible Variants at Position 197 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 TAT TTG GCG TAT GCC GAT ATC GAC TAT GAC CAT-3' EcoRV+ ClaI- Seq ID No 11 which also inserted an EcoRV site (codons 200-201) to replace the ClaI 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 (PstI-SstI 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.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 21 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.

SUBSTITUTE SHEET (RULE 26) TABLE II 0 Synthetic Oligonucleotides Used for Cassette Mutagenesis to Produce M197X Variants C LAAM12 GG GAA GTT TCG AAT GAA AAC G Seq ID No 12 V AAM13 Xl97bs Seq ID No 13 C (EcoRV) GTC GGC ATA TG CAT ATA ATC ATA GTT GCC GTT TTC ATT (BstBI) 4 LAAM14 1197 Seq ID No 14 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG ATC TAT GCC GAC (EcoRV-) m LAAM1 F197 Seq ID No 3: (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG TTC TAT GCC GAC (EcoRV-) m m LAAM16 V197 Seq ID No 16 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG GTT TAT GCC GAC (EcoRV-)

C

m LAAM17 S197 Seq ID No 17 N (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG AGC TAT GCC GAC (EcoRV-)

O

LAAM18 P197 Seq ID No 18 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG CCT TAT GCC GAC (EcoRV-) LAAM19 T197 Seq ID No 19 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG ACA TAT GCC GAC (EcoRV-) Y197 Seq ID No (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG TAC TAT GCC GAC (EcoRV-) LAAM21 H197 Seq ID No 21 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG CAC TAT GCC GAC (EcoRV-) LAAM22 LAAM2 3 LAAM2 4 LAAM2 5 LAAM2 6 LAAM2 7 LAAM2 8 LAAM2 9 G197 (BstBI) Q197 (BstBI) N197 (BstBI) K197 (BstBI) D197 (BstBI) E197 (BstBI) C197 (BstBI) W197 (BstBI) Ri197 (BstBI) CG PAAT CG AAT CG AAT CG AAT CG AAT CG AAT CG AAT CG AAT CG AAT CAA AAC GAA AAC CAA AAC CAA ?AAC GAA AAC CAA AAC CAA AAC GAA AAC GAA AAC

GC

GGC

GGC

GC

GGC

GGC

CGC

GGC

GGC

AAC

AAC

AAC

AAC

AAC

AAC

PIAC

AAC

AAC

TAT CAT TAT CAT TAT GAT TAT GAT TAT GAT TAT GAT TAT CAT TAT GAT TAT GAT

TAT

TAT

TAT

TAT

TAT

TAT

TAT

TAT

TAT

TTG CGC TAT TTG CAA TAT TTG AAC TAT TTG AAA TAT TTG GAT TAT TTG GAA TAT TTG TGT TAT TTG TGG TAT TTG AGA TAT

CC

CC

CC

GCC

GCC

CC

CC

GCC

CC

CAC

CAC

CAC

GAC

GAC

GAC

GAC

CAC

CAC

(EcoRV-) (EcoRV-) (EcoRV-) (EcoRV-) (EcoRV-) (EcoRV-) (EcoRV-) (EcoRV-) (EcoRV-) Seq Seq Seq Seq Seq Seq Seq Seq Seq ID No ID No ID No ID No ID No ID No ID No ID No ID No WO 96/30481 PCTIVS96/04029 24 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 NsiI site, thus transformants derived from this strand could be eliminated by screening for the presence of the unique NsiI 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. 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 10il of amylase to 790 41 of the substrate in a cuvette at 25 0 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.

SUBSTITUTE SHEET (RULE 26) WO96/30481 PCT/US96/04029 25 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 calcium chloride and activity is measured as the time taken to give a negative iodine test upon incubation at 30 0 C. Activity is recorded in liquefons per gram or ml (LU) calculated according to the formula: LU/ml or LU/g 570 Vx t x D Where LU=liquefon unit V=volume of sample t=dextrinization time (minutes) D=dilution factor=dilution volume/ml or g of added enzyme.

ALPHA-AMYLASE

Spezyme® AA20 A4 form (A4 form) M197T (A4 form) M197T M197A (A4 form) M197C M197L (A4 form) TABLE III SPECIFIC ACTIVITY (as of AA20 value) on: Soluble Substrate Starch 100 100 105 115 93 94 85 103 75 83 62 81 88 89 85 51 17 Example 4 Characterization of Variant Variant M15L made as per the prior examples did not show increased amylase activity (Table III) and was still inactivated by hydrogen peroxide (Fig. It did, however, show significantly increased SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 26 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

CO

3 Typical liquefaction conditions were: Starch 32%-35% solids Calcium 40-50 ppm (30 ppm 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 0 -107 0 C. Samples of starch were transferred from the jet cooker to a 95C 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.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 27 TABLE IV Performance of Variants M15L (A4 form) and M15L in Starch Liquefaction pH DE after 90 Mins.

Spezyme® AA20 5.9 9.9 (A4 form) 5.9 10.4 Spezyme® AA20 5.2 1.2 (A4 form) 5.2 2.2 Spezyme® AA20 Spezyme® AA20 Spezyme® AA20 5.9 5.9 5.5 5.5 5.2 5.2 9.3* 11.3* 3.25** 6.7** 0.7** 3.65** average of three experiments average of two experiments Example Construction of M15X Variants Following generally the processes described in Example 3 above, all variants at M15 (M15X) were produced in native B. licheniformis 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 BstBl site at codons 11-13 and a Mscl site at codons 18-20 were introduced using the two oligonucleotides shown below: 5'-G ATG CAG TAT TTC GAA CTGG TAT A-3' BstBl Seq ID No 48 Seq ID No 49 5'-TG CCC AAT GAT GGC CAA CAT TGG AAG-3' Mscl 2) The vector for M15X cassette mutagenesis was then constructed by subcloning the Sfil-SstII fragment from the mutagenized amylase (BstBl+, Mscl+) into plasmid pBLapr. The resulting plasmid was SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 28 then digested with BstBl 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 SnaB1 site allowing for the transformants derived from the bottom strand to be eliminated by screening for the SnaB1 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.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 29 TABLE V Synthetic Oligonucleotides Used for Cassette Mutagenesis to Produce M15X Variants (BstBl) C GAA TGG TAT GCT CCC AAT GAC GG (Mscl) Seq ID No (BstBl) C GAA TGG TAT CGC CCC AAT GAC GG (Mscl) Seq ID No 51 (BstBl) C GAA TGG TAT AAT CCC AAT GAC GG (Mscl) Seq ID No 52 (BstBl) C GAA TGG TAT GAT CCC AAT GAC GG (Mscl) Seq ID No 53 (BstBl) C GAA TGG TAT CAC CCC AAT GAC GG (Mscl) Seq ID No 54 (BstBl) C GAA TGG TAT AAA CCC AAT GAC GG (Mscl) Seq ID No (BstBl) C GAA TGG TAT CCG CCC AAT GAC GG (Mscl) Seq ID No 56 (BstBl) C GAA TGG TAT TCT CCC AAT GAC GG (Mscl) Seq ID No 57 (BstBl) C GAA TGG TAC ACT CCC AAT GAC GG (Mscl) Seq ID No 58 (BstBl) C GAA TGG TAT GTT CCC AAT GAC GG (Mscl) Seq ID No 59 (BstBl) C GAPA TGG TAT TGT CCC AAT GAC GG (Mscl) Seq ID No (BstBl) C GAA TGG TAT CAA CCC AAT GAC, GG (Mscl) Seq ID No 61 (BstBl) C GAA TGG TAT GAA CCC AAT GAC GG (Mscl) Seq ID No 62 (BstBl) C GAA TGG TAT GGT CCC AAT GAC GG (Mscl) Seq ID No 63 M1IT (BstBl) C GAA TGG TAT ATT CCC AAT GAC GG (Msci) Seq ID No 64 (BstBi) C GA7X TGG TAT TTT CCC AAT GAC GG (Msci) Seq ID No (BstBi) C GAA TGG TAC TGG CCC AAT GAC GG (Msci) Seq ID No 66 (BstBi) C GAA TGG TAT TAT CCC AAT GAC GG (Mscl) Seq ID No 67 (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 Conservative substitutions were made in some cases to prevent introduction of new restriction sites.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 30 Example 6 Bench Liquefaction with M15X Variants Eleven alpha-amylase variants with substitutions for M15 made as per Example 5 were assayed for activity, as compared to Spezyme® (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 l05-106 0

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 mi/min. The starch was recovered from the end of the coil and was transferred to the secondary hold (95 0

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 95C for 5 minutes in 10mM 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 31 be a broad correlation between expression of amylase activity and thermal stability. The licheniformis 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. 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 T M (a protease, commercially available from Novo Industries, of the type commonly used in ADD) were added to two commercially available bleach-containing ADD's: CascadeTM (Procter and Gamble, Ltd.) and Sunlight TM (Unilever) and the time course of inactivation of the amylase variants and TermamylTM (a thermally stable alpha-amylase available from Novo Nordisk, A/S) followed at 0 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. lla and lib), both forms of the M197T variant were much more stable than Termamyl'M 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 SavinaseTM, SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 32 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 0 C, 60 mins.). The results are shown in Figs. lla and lib.

Example 9 Characterization of M15X Variants All M15X variants were propagated in Bacillus subtilis 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 amylases have specific activities greater than that of Spezyme® A benchtop heat stability assay was performed on the variants by heating the amylase at 90 0 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 Spezyme® AA20 in this assay. Those variants that exhibited reasonable stability in this assay (reasonable stability defined as those that retained at least about of Spezyme® 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 Spezyme® 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 pHliquefaction performance and/or increased specific activity.

Example 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, Burstein, Y. and SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIS96/04029 -33 Patchornik, A. (1975) Biochemistry 14 (20) 4497-4503). Fig. 17 shows the inactivation of B. licheniformis 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 alphaamylase can be prevented. Conversely, as shown in Fig. 18, at pH 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 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-Amylase Double Mutants W138 and M197 Certain variants of W138 Y and A) were made as double mutants, with M197T (made as per the disclosure of Example 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.

licheniformis 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 Phenylalanine 133 134 135 136 137 138 139 140 141 142 143 CAC CTA ATT AAA GCT TTC ACA CAT TTT CAT TTT Seq ID No 42 Hind III Tryptophan 138 to Tyrosine 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 34 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 GCA 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 SstII/SstI 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 Y, L, H and C) were made.

The 1.24kb Asp718-SstI 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 35 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 Cysteine CC GGA GAA CAC CTA ATT AAA GCC TGC 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 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- SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 36 23 0 C) for about 30 min. (non-heated). A second set of samples was warmed from room temperature to about 65 0 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. licheniformis 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 0 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.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 37 Table VI Reducing Sugar Enzyme Concentration Standard Deviation No Enzyme 1.64 0.12 Wild-Type 4.97 0.30 M15S/M197T 5.40 0.36 M15T/M197T 5.85 0.38 W138Y/M197T 6.48 0.36 M15S/W138Y/M197T 6.04 0.74 M15T/W138Y/M197T 6.27 0.49 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 0 C. Dishes were loaded in an ASKO Model 770 dishwasher and washed at 45 0 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.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 38 Table VII Soil Removed Enzyme (Average of All Dishes) Wild-Type 61 M15S/M197T 66 M15T/M197T 71 W138Y/M197T 68 M15S/W138Y/M197T 62 M15T/W138Y/M197T 72 The data show that the mutant enzymes provided a benefit greater than that provided by the wild-type. Wild-type amylase provided a greater cleaning benefit in removing oatmeal than did ADD without amylase.

Example 12 Dish Care Cleaning Composition 1% 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% TAED were added. Samples of these formulations were placed at room temperature (21-23 0 C) or at 38 0 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 0 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 39 TAED and they provide a significant performance benefit in removing starchy food stains in the wash.

Example 13 Oxidatively Sable Protease/ Oxidatively Stable Amylase Stability Studies Enzyme granules containing either: 1) wild-type protease and wildtype amylase; or 2) bleach stable protease (GG36-M222S) made by the methods described in US Re 34606 and bleach stable amylase 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 0 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 WT Amylase 104 Water WT Protease 94 Water M222S Protease 119 Water TYT Amylase 88 3% Peroxide WT Amylase 14 3% Peroxide WT Protease 7 3% Peroxide M222S Protease 116 3% Peroxide TYT Amylase 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 40 enzymes loses over 80% of its initial activity in the same period of time.

Example 14 Comparative Activity Profile of Amylase Variants The activity profiles of amylase variants were obtained. Two Phadebas tablets (Phadebas Amylase Test Kit, Pharmacia Diagnostics) were dissolved in separate vials of 12 milliliters of buffer (0.05M borate/0.05M potassium phosphate/0.005M calcium chloride) at pH-of about 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0 to determine relative starch hydrolysis activity of each tested enzyme. After addition of the two tablets, the resultant pH values were 6.21, 7.0, 7.68, 8.75, 9.72 and 10.63. Each vial was mixed by magnetic stirring and, while mixing, 200 microliter aliquots were pipetted into a 96 well Costar polystyrene plate. Enzyme samples were obtained from solutions containing 9.9 mg/ml of Termamyl® (wild type Bacillus licheniformis alpha amylase available from Novo Nordisk, Denmark), 5.2 mg/ml Spezyme® AA20 (Bacillus licheniformis alpha amylase available from Genencor International, Inc., South San Francisco Ca.) or 4.1 mg/ml mutant amylase according to the invention (M15T/W138Y/M197T). Each sample was diluted to 1/5000 into an amylase assay buffer (50 mM acetate buffer, pH 6.7, 5mM CaCl 2 .002% Tween 20. Ten microliters of diluted enzyme were added to the substrate solution by multichannel pipette. The reaction mixture was mixed at 1100 rpm using an Ika-Schuttler MTS 2 plate shaker for 30 minutes. The reaction was terminated by filtering insoluble substrate particles from supernatant containing enzyme and solubilized blue dye using 0.45 micron hydrophilic 96 well filtration plates (available in the Multiscreen Filtration System from Millipore, Bedford, Mass.). The samples were removed with a multichannel pipette. A 200 microliter control was used for each pH containing substrate with no enzyme.

From the filtered supernatant, 100 microliter aliquots of each sample were drawn into another 96 well Costar polystyrene plate, again by multichannel pipette, and the absorbance was read at 620nm. The results are shown in Figure 23 by plotting percent SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 41 activity vs. incubation time. As shown in Figure 23, the mutant enzyme according to the present invention possesses superior activity at a pH range of from at least 6 to about 8.5 when compared to Termamyl® or Example Oxidative Stability of Amylase to Peracetic Acid 12.3 microliters of amylase was diluted into an Eppendorf tube containing 977.7 microliters of a solution of 0.1M CHES (2-ncyclohexylaminoethane-sulfonic acid) and 0.005M calcium chloride at a pH of 10.0 at 25°C. The sample was mixed by vortexing and a microliter aliquot was removed to determine initial activity determination on maltoheptaoside. The remaining 980 microliters were combined with 10 microliters of 32% peracetic acid. During the test, the samples were incubated in a heating block with glycerol as the conducting fluid. The temperature was 52°C as measured inside of the reaction tube. The caps of the tubes were sealed during the reaction. The pH of the buffer at 52 0 C was approximately 9.3, a value consistent with the expected pH shift for CHES buffer at higher temperature as reported in Methods of Enzymology, volume 87 (1982).

The sample was vortexed and 10 milliliter aliquots removed for activity determination at 0, 15, 30, 45 and 60 minutes. The results are shown in Figure 24 by plotting the relative activity (based on the release of hydrolyzed starch into solution) vs. pH.

As shown in Figure 24, the mutant enzyme according to the present invention retains an exceptionally high percentage of its initial activity when compared to Termamyl® when incubated with an oxidant at a ph of 9.3.

Example 16 Comparative Wash Performance of Amylase in Liquid Laundry Detergent The wash performance ability of mutant amylase according to the invention in typical liquid laundry detergent conditions in comparison with commercially available wild type amylase was tested. Corn starch/india ink soiled cotton swatches PEPD 7435WRL SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIS96/04029 42 (obtained from Scientific Services S/D, Inc., Sparrow Bush, N.Y.) were used for testing wash performance of amylase additives.

Grease releasing TIDE® concentrated detergent (available commercially and from Proctor Gamble, Cincinnati Ohio) was heated at 90°C for one hour to inactivate any enzymes present protease or lipase). Amylases used were the Termamyl® 60T amylase (wild type Bacillus licheniformis amylase available from Novo Nordisk, Denmark) and the inventive amylase, M15T/W138Y/M197T.

gram aliquots of liquid detergent and amylase sufficient to result in 0.05, 0.1, 0.5, or 1.0 ppm of amylase in the final wash solution (either Termamyl® or M15T/W138Y/M197T) were measured and set aside. A control was performed with no amylase added.

Wash tests were performed in a Model 7243S Terg-o-tometer (United States Testing Co., Hoboken, The wash liquor comprised distilled water with 10 ml of hard water concentrate added to result in a final concentration of 100 ppm Ca 2 ion and 50 ppm Mg 2 One liter of wash liquor was added to each Terg-o-tometer pot.

When the wash liquor reached the appropriate temperature, the agitators were turned on and the detergent and amylase were simultaneously added to the wash liquor. After allowing 3 minutes for dissolution, the swatches were added to the wash liquor. After minutes of agitation at 100rpm, the agitators were turned off and the swatches removed from the Terg-o-tometer pots and rinsed in a washing machine with cold water for 10 minutes. After rinsing, the starch swatches were removed and pressed dry. The swatches were then read with a reflectometer to determine the amount of cleaning. Trials were performed at both 40*C and 55*C. The results are shown in Figures 25 and 26, respectively, plotting the change in reflectance (normalized for detergent alone) vs. ppm amylase added.

As shown in Figures 25 and 26, the mutant amylase of the present invention significantly outperforms commercially available amylase in cleaning ability.

Example 17 Wash Performance of Inventive Amylase in Liquid Detergents SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 43 The wash performance of mutant amylase according to the present invention was tested in combination with two commercially available liquid detergent compositions, SA8@ (commercially available from Amway Corp., Ada, Michigan) and Purex® (commercially available from the Dial Corp., Phoenix, Arizona).

Mutant amylase (M15T/W138Y/M197T) was added to final concentrations in the wash liquor of 0.0, 0.05, 0.1, 0.5, 1.0 and 5.0 ppm. SA8® was added to the wash liquor in a quantity of 0.75gram/liter to bring the wash solution to a pH of 6.5 and a temperature of Purex® was added to the wash liquor in a quantity of gram/liter to bring the wash solution to a pH of 9.0 and a temperature of 40°C. Wash procedure was otherwise as in Example 16. The swatches were measured to determine the change in reflectance (normalized for detergent alone). The results are given in Figure 27.

As can be seen in Figure 27, the addition of amylase significantly improved the cleaning ability of the detergents.

SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 44 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: GENENCOR INTERNATIONAL, INC.

(ii) TITLE OF INVENTION: An Improved Laundry Detergent Composition Comprising Amylase (iii) NUMBER OF SEQUENCES: 68 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Genencor International STREET: 180 Kimball Way CITY: South San Francisco STATE: CA COUNTRY: USA ZIP: 94080 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Stone, Christopher L.

REGISTRATION NUMBER: 35,696 REFERENCE/DOCKET NUMBER: GC220-4 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (415) 742-7555 TELEFAX: (415) 742-7217 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 56 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GATCAAAACA TAAAAAACCG GCCTTGGCCC CGCCGGTTTT TTATTATTTT TGAGCT 56 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TGGGACGCTG GCGCAGTACT TTGAATGGT 29 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGATGCAGTA CTTTGAATGG TACCTGCCCA ATGA 34 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GATTATTTGT TGTATGCCGA TATCGACTAT GACCAT 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGGGGAAGGA GGCCTTTACG GTAGCT 26 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCGGCTATGA CTTAAGGAAA TTGC 24 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 46 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTACGGGGAT GCATACGGGA CGA 23 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTACGGGGAT TACTACGGGA CCAAGGGAGA CTCCC INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCGGTGGGGC CAAGCGGGCC TATGTTGGCC GGCAAA 36 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CATCAGCGTC CCATTAAGAT TTGCAGCCTG CGCAGACATG TTGCT. INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 47 LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GATTATTTGG CGTATGCCGA TATCGACTAT GACCAT 36 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAAGTTTC GAATGAAAAC G 21 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTCGGCATAT GCATATAATC ATAGTTGCCG TTTTCATT 38 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CGAATGAAAA CGGCAACTAT GATTATTTGA TCTATGCCGA C 41 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 48 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGAAAA CGGCAACTAT GATTATTTGT TCTATGCCGA C 41 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: CGAATGAAAA CGGCAACTAT GATTATTTGG TTTATGCCGA C 41 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGAATGAAAA CGGCAACTAT GATTATTTGA GCTATGCCGA C 41 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGAATGAAAA CGGCAACTAT GATTATTTGC CTTATGCCGA C 41 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 49 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGAAAA CGGCAACTAT GATTATTTGT ACTATGCCGA C 41 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGAATGAAAA CGGCAACTAT GATTATTTGC ACTATGCCGA C 41 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CGAATGAAAA CGGCAACTAT GATTATTTGG GCTATGCCGA C 41 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CGAATGAAAA CGGCAACTAT GATTATTTGC AATATGCCGA C 41 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 50 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CGAATGAAAA CGGCAACTAT GATTATTTGA ACTATGCCGA C 41 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GCAATGAAAA CGGCAACTAT GATTATTTGA AATATGCCGA C 41 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CGAATGAAAA CGGCAACTAT GATTATTTGG ATTATGCCGA C 41 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CGAATGAAAA CGGCAACTAT GATTATTTGG AATATGCCGA C 41 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 51 LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CGAATGAAAA CGGCAACTAT GATTATTTGT GTATTGCCGA C 41 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CGAATGAAAA CGGCAACTAT GATTATTTGT GGTATGCCGA C 41 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGAAAA CGGCAACTAT GATTATTTGA GATATGCCGA C 41 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 1968 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AGCTTGAAGA AGTGAAGAAG CAGAGAGGCT ATTGAATAAA TGAGTAGAAA GCGCCATATC GGCGCTTTTC TTTTGGAAGA AAATATAGGG AAAATGGTAC TTGTTAAAAA TTCGGAATAT 120 TTATACAACA TCATATGTTT CACATTGAAA GGGGAGGAGA ATCATGAAAC AACAAAAACG 180 GCTTTACGCC CGATTGCTGA CGCTGTTATT TGCGCTCATC TTCTTGCTGC CTCATTCTGC 240 AGCAGCGGCG GCAAATCTTA ATGGGACGCT GATGCAGTAT TTTGAATGGT ACATGCCCAA 300 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 52

TGACGGCC.AP

TACTGCCGTC

TGCTTACGAC

CGGCACAAAA

TTACGGGGAT

GGTTGAAGTC

CTGGACACAT

GTACCATTTT

TCAAGGAAAG

GTATGCCGAC

TTGGTATGCC

ATTTTCTTTT

TACGGTAGCT

AAATTTTAAT

ACAGGGAGGC

GTTGAAATCG

GACTGTCCAA

ATACCCTCAG

TCCTGCCTTG

AGCACAGCAT

CTCGGTTGCA

A.ATGTATGTC

GGAGCCGGTT

TTCAATTTAT

TTTTTATTTT

GTGTCATCAG

TGAAATGGCA

GCGGGTGATC

CATTGGAAGC

TGGATTCCCC

CTTTATGATT

GGAGAGCTGC

GTGGTCATCA

GATCCCGCTG

TTTCATTTTC

GACGGAACCG

GCTTGGGATT

ATCGATTATG

AATGAACTGC

TTGCGGGATT

GAATATTGGC

CATTCAGTGT

GGCTATGATA

GTTACATTTG

ACATGGTTTA

GTTTTCTACG

AAACACAAAA

GATTATTTCG

AATTCAGGTT

GGCCGGCAAA

GTCATCAATT

GTTCAAAGAT

GCCCGTCTTA

CCCTCAGGAA

ACGTTATCTG

AATCATCCTG

GTTTGCAAAA

CGGCATATAA

TAGGGGAGTT

AATCTGCGAT

ACCACAAAGG

ACCGCAACCG

CGGGGCGCGG

ATTGGGACGA

GGGAAGTTTC

ACCATCCTGA

AATTGGACGG

GGGTTAATCA

AGAATGACTT

TTGACGTGCC

TGAGGAAATT

TCGATAACCA

AGCCGCTTGC

GGGATATGTA

TTGAACCGAT

AC CAC CAT GA

TGGCGGCATT

ACGCCGGTGA

CGGAAGGCTG

AGAAGAGCAG

TAAATTTCTT

GGACTTGCTG

ATGTAGCAAA

AGACTGTGAC

CGACTCGGCA TATTTGGCTG GGGAACGAGC CAAGCGGATG TCATCAAAAA GGGACGGTTC CAAAAGTCTT CATTCCCGCG CGGCGCTGAT GCGACCGAAG CGTAATTTCA GGAGAACACC CAGCACATAC AGCGATTTTA GTCCCGAAAG CTGAACCGCA CAATGAAAAC GGCAACTATG TGTCGCAGCA GAAATTAAGA TTTCCGTCTT GATGCTGTCA TGTCAGGGAA AAAACGGGGA GGGCGCGCTG GAAAACTATT GCTTCATTAT CAGTTCCATG GCTGAACGGT ACGGTCGTTT TGATACACAG CCGGGGCAAT TTACGCTTTT ATTCTCACAA CGGGACGAAA GGAGACTCCC CTT.AAAAGCG AGAAAACAGT CATTGTCGGC TGGACAAGGG AATAACAGAC GGACCCGGTG GACATGGCAT GACATTACCG GGGAGAGTTT CACGTAAACG AGAGGACGGA TTTCCTGAAG TGATTACATT TTATAATTAA ACAGTTTGAA TCGCATAGGT GAAAGCAAAT GTGTCGAAAA GGATGAATTG AAAAAGCT

AACACGGTAT

TGGGCTACGG

GGACAAAGTA

ACATTAACGT

ATGTAACCGC

TAATTAAAGC

AATGGCATTG

TCTATAAGTT

ATTATTTGAT

GATGGGGCAC

AACACATTAA

AGGAAATGTT

TGAACAAAAC

CTGCATCGAC

CCAAGCATCC

CGCTTGAGTC

GGGAATCTGG

AGCGCGAAAT

ATGCGTACGG

AAGGCGACAG

GGGCAAAGCG

GAAACCGTTC

GCGGGTCGGT

GAAATCCGTT

TTTTAACAAA

AAGGCGGGGA

TGACGGTATC

360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1968 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 483 amino acids TYPE: amino acid STP.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein SUBSTITUTE SHEET (RULE 26) WO 96/30481 WO 9630481PCTIUS96/04029 53 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Ala Asn Leu Asn Gly Thr Leu Met Gin Asn Ala Thr Gi y Gi y Val Glu Ile Gi y 145 Asp Phe Tyr Al a Leu 225 Leu Phe Tyr His Arg 305 Vai Ser Asp Giu Ser Giu Giu Tyr Asp Ser 130 Arg Gi y Gin Asp Al a 210 Asp Arg Thr Leu Tyr 290 Lys Thr Thr Gly Gin His Giy Gin Ala Phe His Leu Gin Gly Asp 100 Vai Thr 115 Gly Giu Gly Ser Thr Asp Giy Lys 180 Tyr Leu 195 Giu Ile Giy Phe Asp Trp Vai Ala 260 Asn Lys 275 Gin Phe Leu Leu Phe Val Vai Gin 340 His Ile Asp Gin Ser Vai Al a His Thr Trp 165 Al a Met Lys Arg Vai 245 Glu Thr His Asn Asp 325 Thr Lys Al a Gi y 55 Giy Ile.

Ile Giu Ile 135 Ser Giu Asp Al a Trp 215 Asp His Trp Phe Al a 295 Thr His Phe Arg Leu 25 Vai Trp 40 Tyr Giy Thr Val Lys Ser Asn His 105 Vai Asp 120 Lys Ala Asp Phe Ser Arg Trp Giu 185 Asp Ile 200 Giy Thr Ala Vai Val Arg Gin Asn 265 Asn His 280 Ser Thr Vai Vai Asp Thr Lys Pro 345 Tyr Phe Gin Asn Ile Pro Ala Tyr Arg Thr 75 Leu His 90 Lys Giy Pro Ala Trp, Thr Lys Trp 155 Lys Leu 170 Vai Ser Asp Tyr Trp, Tyr Lys His 235 Giu Lys 250 Asp Leu Ser Vai Gin Giy Ser Lys 315 Gin Pro 330 Leu Ala Glu Trp Tyr Asp Pro Asp Lys Ser Gi y Asp His 140 His Asn Asn Asp Ala 220 Ile Thr Gi y Phe Gi y 300 His Gi y Tyr Ser Al a Leu Tyr Arg Al a Arg 125 Phe Trp Arg Giu His 205 Asn Lys Gi y Al a Asp 285 Gly Pro Gin Al a Al a Tyr Tyr Gi y Asp Asp 110 Asn His Tyr Ile Asn 190 Pro Giu Phe Lys Leu 270 Val Tyr Leu Ser Phe 350 Met Pro Tyr Leu Lys Gly Asp Leu Thr Lys Ile Asn Ala Thr Arg Val Phe Pro His Phe 160 Tyr Lys 175 Gly Asn Asp Val Leu Gin Ser Phe 240 Giu Met 255 Glu Asn Pro Leu Asp Met Lys Ser 320 Leu Glu 335 Ile Leu SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 54 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 Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gin Tyr Ala Tyr Gly Ala Gin His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile 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 Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gin Arg INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 511 amino acids TYPE: amino acid STRANDEDNESS: single 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 Ala Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu 25 Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro Asn Asp Gly 40 His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu Ala Glu His Gly 55 Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gin Ala 70 75 Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe His 90 Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu Gin 100 105 110 Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn Val Tyr Gly Asp 115 120 125 Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp Val Thr 130 135 140 SUBSTITUTE SHEET (RULE 26) WO 96/30481 WO 9630481PCT1US96/04029 Ala 145 His Thr Trp Al a Met 225 Lys Arg Val Glu Thr 305 His Asn Asp Thr Gi y 385 Ser Lys His Asn Arg 465 Thr Glu Val Leu Tyr Asp Trp 210 Tyr Arg Leu Asn Tyr 290 Asn Al a Gi y Asn Trp 370 Tyr Gin Al a His S er 450 Met Gi y Phe Ala Asp Thr His Trp His Leu Asn 200 Ser Asn 215 Tyr Asp Tyr Ala His Ile Lys Thr 280 Leu Gly 295 Val Phe Gly Gly Lys His Pro Gly 360 Ala Tyr 375 Tyr Gly Ala Leu Ala Tyr Trp Thr 440 Leu Ile 455 Gln Asn Pro Val Gly Ser Arg Phe Trp 185 Ar g Glu His Asn Lys 265 Gl y Al a Asp Gi y Pro 345 Gln Ala Asp Lys Gi y 425 Arg Thr Ala Val Val Glu 160 Ser Asp Lys Leu Ile 240 Phe Trp Al1 a Lys Phe 320 Leu Val Gln Ser Asp 400 Leu Asp Al a Lys Ile 480 Gi y SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 56 500 505 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 520 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Met Phe Thr Tyr Glu Tyr Tyr Gi y Asn Asp 145 Asn Arg Tyr Ile Ser 225 Asp Al a Ile Arg Gly Arg Gly Asn Met Ile Gin Arg Lys Thr His Lys Asp Thr Val 130 Al a Gin Phe His Phe 210 Giu His Asn Lys Leu Thr Pro Leu Gi y Leu Lys 115 Gin Thr Giu Pro Phe 195 Lys Asn Pro Giu Phe Val Ser Asn Ser Leu Gi y 100 Ser Val Giu Thr Gi y 180 Asp Phe Gi y Asp Leu 260 Ser Leu Al a Asp Asp Ser Giu Giu Tyr Asp Ser 165 Arg Gi y Arg Asn Val 245 Ser Phe Met Val Gi y Ile 70 Gin Phe Leu Gi y Val 150 Giu Gi y Ala Gi y Tyr 230 Val Leu Leu C ys Asn Gin 55 Gi y S er Gin Gin Asp 135 Thr Giu Asn Asp Giu 215 Asp Al a Asp Arg Thr Gi y 40 His Ile Asp Gin Asp 120 Val Al a Tyr Thr Trp 200 Gi y Tyr Giu Gi y Asp Leu 25 Thr Trp Thr Asn Lys 105 Al a Val Val Gin Tyr 185 Asp Lys Leu Thr Phe 265 Trp Lys Leu Leu Lys Al a Gi y 90 Gi y Ile Leu Glu Ile 170 Ser Glu Al a Met Lys 250 Arg Val Phe Met Arg Val 75 Tyr Thr Gi y Asn Vai 155 Lys Asp Ser Trp Tyr 235 Lys Ile Gin Val Ser Gin Tyr Leu Gin Trp Ile Gly Pro Val Arg Ser Leu 125 His Lys 140 Asn Pro Ala Trp Phe Lys Arg Lys 205 Asp Trp 220 Ala Asp Trp Gly Asp Ala Ala Val Leu Phe Asn Pro Tyr Thr 110 His Aila Al a Thr Trp 190 Ile Giu Val Ile Al a 270 Arg Pro Giu Asp Pro Asp Lys Ser Gi y Asn Asp 175 His Ser Val Asp Trp 255 Lys Gin Arg Lys Arg Thr Val S er Ile Trp Ala Ala Leu Tyr Arg Al a Arg 160 Phe Trp Arg Ser Tyr 240 Tyr His Aa SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 57 Thr Gly 305 Phe Gly His Gly Tyr 385 Gly Leu Tyr Thr Ile 465 Asn Val Gly 290 Lys Asp Gly Pro Gin 370 Ala Asp Lys Gly Arg 450 Thr Ala Lys 275 Lys Leu Val Tyr Glu 355 Ser Phe Met Asp Pro 435 Glu Asp Gly Ile Glu Glu Pro Asp 340 Lys Leu Ile Tyr Asn 420 Gin Gly Gly Glu Gly 500 Met Asn Leu 325 Met Ala Glu Leu Gly 405 Ile His Asp Pro Thr 485 Ser Phe Tyr 310 His Arg Val Ser Thr 390 Thr Glu Asp Ser Gly 470 Trp Asp Thr 295 Leu Phe Arg Thr Thr 375 Arg Lys Pro Tyr Ser 455 Gly Tyr Gly 280 Val Asn Asn Leu Phe 360 Val Glu Gly Ile Ile 440 Ala Ser Asp Trp Ala Lys Leu Leu 345 Val Gin Ser Thr Leu 425 Asp Ala Lys Ile Gly 505 Glu Tyr Thr Ser 315 Gin Ala 330 Asp Gly Glu Asn Thr Trp Gly Tyr 395 Ser Pro 410 Lys Ala His Pro Lys Ser Arg Met 475 Thr Gly 490 Glu Phe Trp 300 Phe Ala Thr His Phe 380 Pro Lys Arg Asp Gly 460 Tyr Asn His 285 Gin Asn Asn Gin Ser Ser Val Val 350 Asp Thr 365 Lys Pro Gin Val Glu Ile Lys Glu 430 Val Ile 445 Leu Ala Ala Gly Arg Ser Val Asn 510 Asn Ser Gin 335 Ser Gin Leu Phe Pro 415 Tyr Gly Ala Leu Asp 495 Asp Ala Val 320 Gly Arg Pro Ala Tyr 400 Ser Ala Trp Leu Lys 480 Thr Gly Ser Val Ser Ile Tyr Val Gin INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 548 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Val Leu Thr Phe His Arg Ile Ile Arg Lys Gly Trp Met Phe Leu Leu 1 5 10 Ala Phe Leu Leu Thr Ala Ser Leu Phe Cys Pro Thr Gly Arg His Ala 25 Lys Ala Ala Ala Pro Phe Asn Gly Thr Met Met Gin Tyr Phe Glu Trp SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 58 Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Tyr Tyr Gly Gly Asp 145 Asn Asp Tyr Ile Thr 225 Asp Val Ile Thr Asn 305 Phe Gly Gin Ala Ala 385 Asn Lys Asp Thr Met 130 Gly Gin Phe His Tyr 210 Glu His Asn Lys Gly 290 Lys Asp Ala Pro Lys 370 Leu Gly Leu Lys 115 Gin Thr Glu Pro Phe 195 Lys Asn Pro Thr Phe 275 Lys Leu Ala Phe Thr 355 Arg Ser Thr Gly 100 Ala Val Glu Ile Gly 180 Asp Phe Gly Glu Thr 260 Ser Pro His Pro Asp 340 Leu Cys Ser SSer Glu Gin Tyr Trp Ser 165 Arg Gly Arg Asn Val 245 Asn Phe Leu Asn Leu 325 Met Ala Ser Leu 70 SArg SPhe Tyr Ala Val 150 Gly Gly Val Gly Tyr 230 Val Ile Phe Phe Tyr 310 His Arg Val His Arg 390 Gly Ser Asn Leu Asp 135 Asp Thr Asn Asp Ile 215 Asp Thr Asp Pro Thr 295 Ile Asn Thr Thr Gly 375 Gln Ile Asp Gin Gin 120 Val Ala Tyr Thr Trp 200 Gly Tyr Glu Gly Asp 280 Val Thr Lys Leu Phe 360 Arg Glu Thr Val Lys 105 Ala Val Val Gin Tyr 185 Asp Lys Leu Leu Phe 265 Trp Gly Lys Phe Met 345 Val Pro Gly Ala Gly 90 Gly Ile Phe Glu Ile 170 Ser Glu Ala Met Lys 250 Arg Leu Glu Thr Tyr 330 Thr Asp rrp Tyr Leu 75 Tyr Thr Gin Asp Val 155 Gin Ser Ser Trp Tyr 235 Asn Leu Ser Tyr Asn 315 Thr Asn Asn Phe Pro 395 Ser Leu Gly Val Val Arg Ala Ala 125 His Lys 140 Asn Pro Ala Trp Phe Lys Arg Lys 205 Asp Trp 220 Ala Asp Trp Gly Asp Gly Tyr Val 285 Trp Ser 300 Gly Thr Ala Ser Thr Leu His Asp 365 Lys Pro 380 Cys Val Pro Tyr Thr 110 His Gly Ser Thr Trp 190 Leu Glu Leu Lys Leu 270 Arg Tyr Met Lys Met 350 Thr Leu Phe SPro Asp Lys Ala Gly Asp Lys 175 Arg Ser Val Asp Trp 255 Lys Ser Asp Ser Ser 335 Lys Asn Ala Tyr Ala SLeu Tyr Ala Ala Arg 160 Phe Trp Arg Asp Met 240 Tyr His Gin Ile Leu 320 Gly Asp Pro Tyr Gly 400 Phe Ile Leu Thr SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 59 Asp Tyr Tyr Gly Ile Pro Gin Tyr Asn Ile Pro Ser Leu Lys Ser Lys 405 410 415 Ile Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln 420 425 430 His Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly 435 440 445 Val Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly 450 455 460 Ala Gly Arg Ser Lys Trp Met Tyr Val Gly Lys Gin His Ala Gly Lys 465 470 475 480 Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn 485 490 495 Ser Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val 500 505 510 Trp Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr 515 520 525 Thr Arg Pro Trp Thr Gly Glu Phe Val Arg Trp His Glu Pro Arg Leu 530 535 540 Val Ala Trp Pro 545 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 483 amino acids TYPE: amino acid STRANDEDNESS: single 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 Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu 25 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 40 Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 55 Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 70 75 Gly Glu Leu Gin Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 90 Val Tyr Gly Asp Val Val Ile 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 SUBSTITUTE SHEET (RULE 26) WO 96/30481 WO 9630481PCTIUS96/04029 Ile Gi y 145 Asp Phe Tyr Ala Leu 225 Leu Phe Tyr His Arg 305 Val Ser Thr Thr Giu 385 Asp Ser Gi y Trp Giu 465 Val Gly Giu Gly Ser Thr Asp Gly Lys 180 Tyr Leu 195 Giu Ile Gly Phe Asp Trp, Val Ala 260 Asn Lys 275 Gin Phe Leu Leu Phe Val Vai Gin 340 Giu Ser 355 Gly Asp Ile Leu Phe Asp Vai Ala 420 Ala Lys 435 Asp Ile Trp Giy Arg SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 61 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 487 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Ala Ala Ala Ala Ala Asn Leu Asn Gly Thr Leu Met Gin Tyr Phe Glu 1 5 10 Trp Tyr Met Pro Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp 25 Ser Ala Tyr Leu Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro 40 Ala Tyr Lys Gly Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp 55 Leu Tyr Asp Leu Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys 70 75 Tyr Gly Thr Lys Gly Glu Leu Gin Ser Ala Ile Lys Ser Leu His Ser 90 Arg Asp Ile Asn Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly 100 105 110 Ala Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp 115 120 125 Arg Asn Arg Val Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His 130 135 140 Phe His Phe Pro Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His 145 150 155 160 Trp Tyr His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn 165 170 175 Arg Ile Tyr Lys Phe Gin Gly Lys Ala Trp Asp Trp Glu Val Ser Asn 180 185 190 Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp 195 200 205 His Pro Asp Val Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala 210 215 220 Asn Glu Leu Gin Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile 225 230 235 240 Lys Phe Ser Phe Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr 245 250 255 Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gin Asn Asp Leu Gly 260 265 270 Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe 275 280 285 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 62 Asp Val Pro Leu His Tyr 290 Gly 305 Pro Gin Ala Asp Lys 385 Gly Arg Thr Ala Val 465 Tyr Leu Ser Phe Met 370 His Ala Glu Asp Gly 450 Ile Asp Lys Leu Ile 355 Tyr Lys Gin Gly Gly 435 Glu Asn Met Ser Glu 340 Leu Gly Ile His Asp 420 Pro Thr Ser Arg Val 325 Ser Thr Thr Glu Asp 405 Ser Gly Trp Glu Lys 310 Thr Thr Arg Lys Pro 390 Tyr Ser Gly His Gly 470 Gin Phe His Ala Ala 295 Leu Leu Asn Gly Thr 315 Phe Val Asp Asn His 330 Val Gin Thr Trp Phe 345 Glu Ser Gly Tyr Pro 360 Gly Asp Ser Gln Arg 375 Ile Leu Lys Ala Arg 395 Phe Asp His His Asp 410 Val Ala Asn Ser Gly 425 Ala Lys Arg Met Tyr 440 Asp Ile Thr Gly Asn 455 Trp Gly Glu Phe His 475 Ser 300 Val Asp Lys Gin Glu 380 Lys Ile Leu Val Arg 460 Thr Gin Val Ser Thr Gin Pro Leu 350 Val Phe 365 Ile Pro Gin Tyr Val Gly Ala Ala 430 Gly Arg 445 Ser Glu Gly Lys Pro 335 Ala Tyr Ala Ala Trp 415 Leu Gin Pro Gly His 320 Gly Tyr Gly Leu Tyr 400 Thr Ile Asn Val Ser 480 Val Asn Gly Gly Val Ser Ile Tyr Val Gin Arg 485 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) Met 1 Ala SEQUENCE DESCRIPTION: SEQ ID NO:38: Lys Gin Gin Lys Arg Leu Thr Ala Arg Leu Leu Thr Leu Leu Phe 5 10 Leu Ile Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu 25 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 33 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 63 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Gly Lys 25 Ser INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Ala Ala 25 Ala Ala Asn INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Met Arg Ser Lys Thr Leu Trp Ile Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 Ile Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Asn Leu 25 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 64 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CACCTAATTA AAGCTTTCAC ACATTTTCAT TTT 33 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: CACCTAATTA AAGCTTACAC ACATTTTCAT TTT 33 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CCGCGTAATT TCCGGAGAAC ACCTAATTAA AGCCGCAACA CATTTTCATT TTCCCGGGCG CGGCAG 66 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CCGGAGAACA CCTAATTAAA GCCCTAACAC ATTTTCATTT TC 42 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 65 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CCGGAGAACA CCTAATTAAA GCCCACACAC ATTTTCATTT TC 42 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CCGGAGAACA CCTAATTAAA GCCTGCACAC ATTTTCATTT TC 42 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GATGCAGTAT TTCGAACTGG TATA 24 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: TGCCCAATGA TGGCCAACAT TGGAAG 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGGTAT GCTCCCAATG ACGG 24 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTS96/04029 66 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CGAATGGTAT CGCCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CGAATGGTAT AATCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CGAATGGTAT GATCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CGAATGGTAT CACCCCAATG ACGG 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCT/US96/04029 67 LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGGTAT AAACCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGAATGGTAT CCGCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CGAATGGTAT TCTCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CGAATGGTAC ACTCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIUS96/04029 68 (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CGAATGGTAT GTTCCCAATG ACGG 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGGTAT TGTCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: CGAATGGTAT CAACCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: CGAATGGTAT GAACCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTIS96/04029 69 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CGAATGGTAT GGTCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: CGAATGGTAT ATTCCCAATG ACGG 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGAATGGTAT TTTCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CGAATGGTAC TGGCCCAATG ACGG 24 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CGAATGGTAT TATCCCAATG ACGG 24 SUBSTITUTE SHEET (RULE 26) WO 96/30481 PCTfUS96/04029 70 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: CCGTCATTGG GACTACGTAC CATT 24 SUBSTITUTE SHEET (RULE 26)

Claims (9)

1. An improved laundry detergent composition, the improvement comprising adding to the laundry detergent 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 gene by the substitution of a methionine at a position equivalent to M+197 in B.licheniformis alpha-amylase and the substitution of one or more methionine or tryptophan at a position equivalent to M+15 or W+138 in B. licheniformis alpha-amylase, wherein said detergent has a pH of about 6 to 8.

2. An improved laundry detergent composition according to claim 1, wherein the cleaning composition is a liquid composition.

3. An improved laundry detergent composition according to claim 1 or 2, wherein the mutant alpha-amylase is selected from the group consisting of M15T/M197T; M15S/M197T; W138Y/M 97T; M15S/W138Y/M197T and M15T/W138Y/M 197T.

4. An improved laundry detergent composition according to any one of claims 1 to 3, 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 Bacillus amyloliquefaciens protease.

An improved laundry detergent composition according to claim 4, wherein said mutant protease comprises a substitution selected from the group of amino acids consisting of alanine, cysteine and serine.

6. An improved laundry detergent composition according to claim 4 or 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. IC W:\ilona\Sharonsp153226.doc

7. An improved laundry detergent composition according to any one of claims 1 to 6 wherein said laundry detergent contains bleach.

8. An improved laundry detergent composition according to any one of claims 1 to 7, wherein said laundry detergent is granular.

9. An improved laundry detergent composition according to claim 1, substantially as hereinbefore described with reference to the examples. DATED: 23 February, 2000 PHILLIPS ORMONDE FITZPATRICK Attorneys for: GENENCOR INTERNATIONAL, INC. IC W:\llona\Sharon\spl53226.doc