EP0815193A1 - An improved laundry detergent composition comprising amylase - Google Patents

An improved laundry detergent composition comprising amylase

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Publication number
EP0815193A1
EP0815193A1 EP96909854A EP96909854A EP0815193A1 EP 0815193 A1 EP0815193 A1 EP 0815193A1 EP 96909854 A EP96909854 A EP 96909854A EP 96909854 A EP96909854 A EP 96909854A EP 0815193 A1 EP0815193 A1 EP 0815193A1
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EP
European Patent Office
Prior art keywords
gly
amylase
ala
asp
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP96909854A
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German (de)
French (fr)
Inventor
Christopher C. Genencor International BARNETT
Stephen G. Boyer
Colin Mitchinson
Scott D. Power
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Danisco US Inc
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Genencor International Inc
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Publication of EP0815193A1 publication Critical patent/EP0815193A1/en
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase

Definitions

  • 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 alpha- amylase, 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.
  • Alpha-amylases (alpha-1, -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. li cheniformis, B.
  • amyloliquefaciens B. subtilis, or B. stearothermophilus .
  • the preferred enzymes in commercial use have been those from B. li cheniformis because of their heat stability and performance, at least at neutral and mildly alkaline pH's.
  • alpha-amylase is inactivated by hydrogen peroxide and other oxidants at pH's between 4 and 10.5 as described in the examples herein.
  • alpha-amylase enzymes can be used under dramatically different conditions such as both high and low pH conditions, depending on the commercial application.
  • alpha-amylases may be used in the liquefaction of starch, a process preferably performed at a low pH (pH ⁇ 5.5).
  • 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.
  • 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.
  • 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.
  • 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.
  • the mutant results from substituting a different amino acid for one or more methionine residue (s) in the precursor alpha-amylase.
  • the mutants comprise substitution of one or more tryptophan residue alone or in combination with the substitution of one or more methionine residue in the precursor alpha-amylase.
  • Such mutant alpha-amylases in general, are obtained by in vi tro modification of a precursor DNA sequence encoding a naturally occurring or recombinant alpha-amylase to encode the substitution or deletion of one or more amino acid residues in a precursor amino acid sequence.
  • substitution or deletion of one or more amino acid in the amino acid sequence is due to the replacement or deletion of one or more methionine, tryptophan, cysteine, histidine or tyrosine residues in such sequence, most preferably the residue which is changed is a methionine residue.
  • the oxidizable amino acid residues may be replaced by any of the other 20 naturally occurring amino acids.
  • 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) .
  • 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
  • another oxidizable amino acid such as cysteine, methion
  • Preferred laundry detergents comprise mutants comprising the substitution of a methionine residue equivalent to any of the methionine residues found in B. li cheniformi s alpha-amylase (+8, +15, +197, +256, +304, +366 and +438) .
  • the methionine to be replaced is a methionine at a position equivalent to position +197 or +15 in B. li cheniformi s alpha-amylase.
  • Preferred substitute amino acids to replace the methionine at position +197 are alanine ' (A) , isoleucine (I), threonine (T) or cysteine (C) .
  • the preferred substitute amino acids at position +15 are leucine (L) , threonine (T) , asparagine (N) , aspartate (D) , serine (S) , valine (V) and isoleucine (I), although other substitute amino acids not specified above may be useful.
  • Two specifically preferred mutants of the present invention are M197T and M15L.
  • Another embodiment of this invention relates to laundry detergents comprising mutants comprising the substitution of a tryptophan residue equivalent to any of the tryptophan residues found in B. licheniformis alpha-amylase (see Fig. 2).
  • 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.
  • the alpha-amylase mutants included in the laundry detergents of the present invention 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 alpha- amylases according to the invention which has a pH above about 10 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.
  • 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.
  • reduced oxidative stability may be useful in industrial processes that require the rapid and efficient quenching of enzymatic activity.
  • 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 alpha- amylase mutants derived from a Bacill us strain such as B . li cheniformis , B. amyloliquefaciens , and B. stearothermophilus, and most preferably from Bacillus li cheniformis .
  • a laundry detergent comprising a novel form of the alpha-amylase normally produced by B. li cheniformis .
  • This novel form designated as the A4 form, has an additional four alanine residues at the N- terminus of the secreted amylase.
  • Derivatives or mutants of the A4 form of alpha-amylase are encompassed within the present invention.
  • derivatives or mutants of the A4 form it is meant that the present invention comprises the A4 form alpha- amylase containing one or more additional mutations such as, for example, mutation (substitution, replacement or deletion) of one or more oxidizable amino acid(s).
  • a composition embodiment of the present invention comprises laundry detergent compositions, liquid, gel or granular, comprising the alpha-amylase mutants described herein.
  • detergent compositions comprising a +197 position mutant either alone or in combination with other enzymes such as endoglycosidases, cellulases, proteases, Upases or other amylase enzymes.
  • laundry detergent compositions comprising a M15X/W138X/M197X mutant, and most preferably a M15T/W138Y/M197T ("TYT") mutant.
  • the compositions of the present invention may include an alpha-amylase mutant having more than one site-specific mutation.
  • the laundry detergent composition of the present invention is used in a method to clean soiled laundry.
  • Fig. la-lc shows the DNA sequence of the gene for alpha-amylase from B. li cheniformis (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. li cheniformis (NCIB8061), Seq ID No 32.
  • Fig. 3a-3b shows an alignment of primary structures of Ba cill us alpha-amylases.
  • Fig. 4a shows the amino acid sequence of the mature alpha-amylase variant M197T, Seq ID No 36.
  • Fig. 4b shows the amino acid sequence of the A4 form of alpha- amylase from B . li cheniformis NCIB8061, Seq ID No 37. Numbering is from the N-terminus, starting with the four additional alanines .
  • Fig. 5 shows plasmid pA4BL wherein BLAA refers to B. li cheniformi s alpha-amylase gene, PstI to Sstl; Amp R refers to the ampicillin- resistant gene from pBR322; and CAT refers to the Chloramphenicol- resistant gene from pC194.
  • Fig. 6 shows the signal sequence-mature protein junctions for B. 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 (Spezyme® AA20 and M197L (A4 form) with 0.88M H 2 0 2 at pH 5.0, 25°C.
  • Fig. 7b shows inactivation of certain alpha-amylases (Spezyme® AA20, M197T) with 0.88M H 2 0 2 at pH 10.0, 25°C.
  • Fig. 7c shows inactivation of certain alpha-amylases (Spezyme® AA20, M15L) with 0.88M H 2 0 2 at pH 5.0, 25°C.
  • Fig. 8 shows a schematic for the production of M197X cassette mutants.
  • Fig. 9 shows expression of M197X variants.
  • Fig. 10 shows thermal stability of M197X variants at pH 5.0, 5mM CaCl 2 at 95°C for 5 mins.
  • Figs. 11a and lib show inactivation of certain amylases in automatic dish care detergents.
  • Fig. 11a shows the stability of certain amylases in CascadeTM (a commercially available dish care product) at 65°C in the presence or absence of starch.
  • Fig. lib shows the stability of certain amylases in SunlightTM (a commercially available dish care product) at 65°C in the presence or absence of starch.
  • Fig. 12 shows a schematic for the production of M15X cassette mutants.
  • Fig. 13 shows expression of M15X variants.
  • Fig. 14 shows specific activity of M15X variants on soluble starch.
  • Fig. 15 shows heat stability of M15X variants at 90°C, pH 5.0, 5mM CaCl 2 , 5 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. li cheniformis wild-type.
  • Fig. 17 shows the inactivation of B. li cheniformis alpha-amylase (AA20 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. licheniformi s alpha-amylase (AA20 at 0.22 mg/ml) with chloramine-T at pH 4.0 as compared to variants M197A (4.3 mg/ml) and M197L (0.53 mg/ml) .
  • Fig. 19 shows the reaction of B. li cheniformis alpha-amylase (AA20 at 0.75 mg/ml) with chloramine-T at pH 5.0 as compared to double variants M197T/ 138F (0.64 mg/ml) and M197T/W138Y (0.60 mg/ml) .
  • Fig. 20 shows the stability testing results of various alpha- amylase multiple mutants incorporated in automatic dish detergent (ADD) formulations at temperatures from room temperature increased to 65°C.
  • ADD automatic dish detergent
  • Fig. 21 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at room temperature over 0-30 days, as determined by percent activity remaining over time.
  • Fig. 22 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at 38°C (100°F) with 80% relative humidity over 0-30 days.
  • Fig. 23 shows the pH activity profile of certain amylases on a Phadebas substrate at 25 ⁇ 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 ("TYT") 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 ("TYT") 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.
  • amylases used in starch liquefaction may be subject to some form of inactivation due to some activity present in the starch slurry (see commonly owned US applications 07/785,624 and 07/785,623 and US Patent 5,180,669, issued January 19, 1993, incorporated herein by reference) .
  • use of an amylase in the presence of oxidants, such as in bleach- or peracid- containing detergents may result in partial or complete inactivation of the amylase. Therefore, the present invention focuses on altering the oxidative sensitivity of amylases which are added to laundry detergents.
  • the mutant enzymes in the laundry detergents of the present invention may also have an altered pH profile and/or altered thermal stability which may be due to the enhanced oxidative stability of the enzyme at low or high pH's.
  • Alpha-amylase as used herein includes naturally occurring amylases as well as recombinant amylases.
  • Preferred amylases in the present invention are alpha-amylases derived from B. li cheniformis or B. stearothermophil us, including the A4 form of alpha-amylase derived from B. li cheniformis as described herein, as well as fungal alpha- amylases such as those derived from Aspergillus (i.e., A. oryzae and A. ni ger) .
  • Recombinant alpha-amylases refers to an alpha-amylase in which the DNA sequence encoding the naturally occurring alpha-amylase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the alpha-amylase sequence. Suitable modification methods are disclosed herein, and also in commonly owned US Patents 4,760,025 and 5,185,258, the disclosure of which are incorporated herein by reference.
  • the relative sequence homology between B. stearothermophil us and B. li cheniformi s amylase is about 66%, as determined by Holm, L. et al. (1990) Protein Engineering 3_ (3) pp. 181-191.
  • the sequence homology between B . li cheniformi s and B . amyloliquefaci ens 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 amylases or other enzymes. For example, structural homology between fungal amylases and bacterial (Bacill us) 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.
  • amino acid position number refers to the number assigned to the mature Bacill us li cheniformi s alpha-amylase sequence presented in Fig. 2.
  • the invention is not limited to the mutation of this particular mature alpha-amylase [ B. li cheniformis ) but extends to precursor alpha-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in B.
  • a residue (amino acid) of a precursor alpha-amylase is equivalent to a residue of B. li cheniformi s alpha-amylase if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B . li cheniformi s alpha-amylase (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally) .
  • Fig. 3 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 li cheniformis numbering, Hisl05; Arg229; Asp231; His235; Glu261 and Asp328.
  • Expression vector refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host.
  • 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 . subti lis aprE promoter.
  • the vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • plasmid and vector are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present.
  • 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.
  • host strains of the same species or genus from which the alpha-amylase is derived are suitable, such as a Bacill us strain.
  • Bacill us strain Preferably an alpha-amylase negative Bacill us strain (genes deleted) and/or an alpha-amylase and protease deleted Bacill us strain such as Bacillus subtilis strain BG2473
  • 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.
  • 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.
  • 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 between about 5.0 and about 10.0 and most preferably between about 6.0 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.
  • 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 ("TYT”) .
  • Another embodiment of the present invention comprises the combination of the mutant alpha-amylase enzymes described herein in combination with other enzymes (i.e., proteases, lipases, cellulases, etc.), and preferably oxidatively stable proteases.
  • Suitable oxidatively stable proteases include genetically engineered proteases such as those described in US Re 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.).
  • protease mutants oxidatively stable proteases
  • mutants having a substitution for the methionine at a position equivalent to M222 in B. amyloliquefaci ens are described in US Re 34606.
  • Suitable methods for determining "equivalent" positions in other subtilisins are provided in Re 34606, EP 257,446 and USSN 212,291, which are incorporated herein by reference.
  • Example 1 Substitutions for the Methionine Residues in B . li cheniformis Alpha-Amylase
  • the alpha-amylase gene (Fig. 1) was cloned from B. li cheniformi s NCIB8061 obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (Gray, G. et al. (1986) J. Bacteriology 166:635-643) .
  • the 1.72kb Pstl-Sstl fragment, encoding the last three residues of the signal sequence; the entire mature protein and the terminator region was subcloned into M13MP18.
  • a synthetic terminator was added between the Bell and Sstl sites using a synthetic oligonucleotide cassette of the form:
  • Bold letter indicate base changes introduced by oligonucleotide.
  • 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 RF1 's. Positives were confirmed by dideoxy sequencing (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and the Pstl-Sstl fragments for each subcloned into an E. coli vector, plasmid pA4BL.
  • B . subtilis pA4BL is a plasmid able to replicate in E. coli and integrate into the B. subtili s 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 subtili s strain BG2473 was a derivative of 1168 which had been deleted for amylase ( ⁇ a- ⁇ jyE) and two proteases (Aapr, Anpr) (Stahl, M.L. and Ferrari, E., J. Bacter. 158:411-418 and US Patent 5,264,366, incorporated herein by reference) .
  • the sacU32(Hy) (Henner, D.J. et al. (1988) J. Bacter. 170:296-300) mutation was introduced by PBS-1 mediated transduction (Hoch, J.A. (1983) 154:1513-1515) .
  • Example 2 Oxidative Sensitivity of Methionine Variants
  • B. li cheniformi s alpha-amylase such as Spezyme® AA20 (commercially available from Genencor International, Inc.) is inactivated rapidly in the presence of hydrogen peroxide (Fig. 7) .
  • Various methionine variants were expressed in shake-flask cultures of B. subtilis and the crude supernatants purified by ammonium sulphate cuts. The amylase was precipitated from a 20% saturated ammonium sulphate supernatant by raising the ammonium sulphate to 70% saturated, and then resuspended. The variants were then exposed to
  • primer LAAM12 (Table II) was used to introduce another silent restriction site (BstBI) over codons 186-188.
  • 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.
  • the common bottom strand of the cassette contained a frame-shift and encoded a Nsil site, thus transformants derived from this strand could be eliminated by screening for the presence of the unique Nsil site and would not be expected, in any case, to lead to expression of active amylase.
  • 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 lO ⁇ l of amylase to 790 ⁇ l of the substrate in a cuvette at 25°C. Rates of hydrolysis were measured as the rate of change of absorbance at 410nm, after a delay of 75 seconds. The assay was linear up to rates of 0.4 absorption units/min.
  • amylase protein concentration was measured using the standard Bio-Rad assay (Bio-Rad Laboratories) based on the method of Bradford, M. (1976) Anal. Biochem. 72:248) using bovine serum albumin standards.
  • Starch Hydrolysis Assay The standard method for assaying the alpha-amylase activity of Spezyme® AA20 was used. This method is described in detail in Example 1 of USSN 07/785,624, incorporated herein by reference. Native starch forms a blue color with iodine but fails to do so when it is hydrolyzed into shorter dextrin molecules.
  • the substrate is soluble Lintner starch 5gm/liter in phosphate buffer, pH 6.2 (42.5gm/liter potassium dihydrogen phosphate, 3.16gm/liter sodium hydroxide) .
  • the sample is added in 25mM calcium chloride and activity is measured as the time taken to give a negative iodine test upon incubation at 30°C.
  • Activity is recorded in liquefons per gram or ml (LU) calculated according to the formula:
  • V volume of sample (5ml)
  • t dextrinization time (minutes)
  • Starch liquefaction was typically performed using a Hydroheater M 103-M steam jet equipped with a 2.5 liter delay coil behind the mixing chamber and a terminal back pressure valve. Starch was fed to the jet by a Moyno pump and steam was supplied by a 150 psi steam line, reduced to 90-100 psi. Temperature probes were installed just after the Hydroheater jet and just before the back pressure valve.
  • Starch slurry was obtained from a corn wet miller and used within two days. The starch was diluted to the desired solids level with deionized water and the pH of the starch was adjusted with 2% NaOH or saturated Na 2 C0 3 . Typical liquefaction conditions were:
  • Starch was introduced into the jet at about 350 ml/min. The jet temperature was held at 105°-107°C. Samples of starch were transferred from the jet cooker to a 95°C second stage liquefaction and held for 90 minutes .
  • the degree of starch liquefaction was measured immediately after the second stage liquefaction by determining the dextrose equivalence (DE) of the sample and by testing for the presence of raw starch, both according to the methods described in the Standard Analytical Methods of the Member Companies of the Corn Refiners Association, Inc., sixth edition.
  • Starch when treated generally under the conditions given above and at pH 6.0, will yield a liquefied starch with a DE of about 10 and with no raw starch. Results of starch liquefaction tests using mutants of the present invention are provided in Table IV. TABLE IV
  • Example 5 Construction of M15X Variants Following generally the processes described in Example 3 above, all variants at M15 (M15X) were produced in native B. li cheniformi s by cassette mutagenesis, as outlined in Fig. 12:
  • Site directed mutagenesis via primer extension in M13 was used to introduce unique restriction sites flanking the M15 codon to facilitate insertion of a mutagenesis cassette. Specifically, a BstBI site at codons 11-13 and a Mscl site at codons 18-20 were introduced using the two oligonucleotides shown below:
  • Mutagenesis cassettes were created as with the M197X variants. Synthetic oligomers, each encoding a substitution at codon 15, were annealed to a common bottom primer. Upon proper ligation of the cassette to the vector, the Mscl is destroyed allowing for screening of positive transformants by loss of this site.
  • the bottom primer contains an unique SnaBl site allowing for the transformants derived from the bottom strand to be eliminated by screening for the SnaBl site. This primer also contains a frameshift which would also eliminate amylase expression for the mutants derived from the common bottom strand.
  • 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® AA20 (commercially available from Genencor International, Inc.) in liquefaction at pH 5.5 using a bench liquefaction system.
  • the bench scale liquefaction system consisted of a stainless steel coil
  • the coil except for each end, was immersed in a glycerol-water bath equipped with thermostatically controlled heating elements that maintained the bath at 105-106°C.
  • 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
  • the li cheniformis 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.
  • cysteine and threonine are preferred for maximal thermal stability under these conditions whereas alanine and isoleucine are of intermediate stability.
  • other substitutions at position +197 result in lowered thermal stability which may be useful for other applications.
  • different substitutions at +197 may have other beneficial properties, such as altered pH performance profile or altered oxidative stability.
  • the M197C variant was found to inactivate readily by air oxidation but had enhanced thermal stability.
  • both M197T and M197A retained not only high thermal stability (Fig. 10), but also high activity (Table III), while maintaining resistance to inactivation by peroxide at pH 5 to pH 10 (Fig. 7) .
  • Example 8 Stability and Performance in Detergent Formulation The stability of the M197T (A4 form), M197T and M197A (A4 form) was measured in automatic dish care detergent (ADD) matrices. 2ppm
  • SavinaseTM (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 SunlightTM (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 65°C.
  • the concentration of ADD product used in both cases was equivalent to 'pre-soak' conditions: 14gm product per liter of water (7 grams per gallon hardness) .
  • Figs. 11a and lib both forms of the M197T variant were much more stable than
  • TermamylTM 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, prewarmed in a test tube and, after vortexing, activities were assayed as a function of time, using the soluble substrate assay.
  • the "+ starch” tube had spaghetti starch baked onto the sides (140°C, 60 mins.). The results are shown in Figs. 11a and lib.
  • Example 9 Characterization of M15X Variants All M15X variants were propagated in Bacillus 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 M15X amylases have specific activities greater than that of Spezyme® AA20. A benchtop heat stability assay was performed on the variants by heating the amylase at 90°C for 5 min. in 50 mM acetate buffer pH 5 in the presence of 5 mM CaCl 2 (Fig. 15) . Most of the variants performed as well as Spezyme® AA20 in this assay.
  • Chloramine-T sodium N-chloro-p-toluenesulfonimide
  • chloramine-T will modify both methionine and tryptophan (Schechter, Y., Burstein, Y. and Patchornik, A. (1975) Biochemistry 14 (20) 4497-4503) .
  • Fig. 17 shows the inactivation of B.
  • tryptophan residue is also implicated in the chloramine-T mediated inactivation event.
  • tryptic mapping and subsequent amino acid sequencing indicated that the tryptophan at position 138 was oxidized by chloramine-T (data not shown) .
  • site-directed mutants were made at tryptophan 138 as provided below:
  • W138 and Ml97 Certain variants of W138 (F, Y and A) were made as double mutants, with M197T (made as per the disclosure of Example 3) .
  • the double mutants were made following the methods described in Examples 1 and 3.
  • single negative strands of DNA were prepared from an M13MP18 clone of the 1.72kb coding sequence (Pst I-Sst I) of the B. li cheniformis 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.
  • the 1.24kb Asp718-Sstl fragment containing the M197T mutation in plasmid pBLapr (W138X, M197T) of Example 7 was replaced by the wild-type fragment with methionine at 197, resulting in pBLapr (W138F), pBLapr (W138Y) and pBLapr (BspE I, Xma I) .
  • mutants 138L, W138H and 138C were made by ligating synthetic cassettes into the pBLapr (BspE I, Xma I) vector using the following primers:
  • a 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg.
  • Certain amylase mutants described above were added to this ADD solution to yield a rate of 0.4 when assayed by the Ceralpha method (Megazyme (Austr.) Pty. Ltd., Parramatta, NSW, Australia) .
  • One set of samples was held at room temperature (21- 23°C) for about 30 min. (non-heated) .
  • a second set of samples was warmed from room temperature to about 65°C after addition of the enzyme (heated) .
  • 30 min. after addition of the enzyme the activity of the amylase mutants was measured and the activity relative to the activity at the time of addition of the enzyme was calculated (relative activity %).
  • a 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg and three cooked pieces of elbow macaroni were added.
  • the amylase mutants described above were added to this ADD solution to yield a final concentration of 5 ppm active enzyme.
  • the tubes were incubated at 50°C for about 30 min. and the concentration of reducing sugars released was measured against a glucose standard curve using the dinitrosalicylic acid method. Results are shown in Table VI.
  • Dishes were evenly soiled with a cooked, blended oatmeal paste and dried overnight at 37°C. Dishes were loaded in an ASKO Model 770 dishwasher and washed at 45°C on the Quick Wash cycle using 10 g of automatic dishwashing detergent containing 5% perborate, 3% TAED and 11 mg of certain amylase enzyme(s) . The plates were weighed before soiling, after soiling and after washing, and the average % soil removed from all plates was calculated. The data are shown below in Table VII.
  • mutant enzymes provided a benefit greater than that provided by the wild-type.
  • Wild-type amylase provided a 20% greater cleaning benefit in removing oatmeal than did ADD without amylase.
  • Example 12 Dish Care Cleaning Composition 1% (w/w) granules of wild-type and mutant amylases were formulated with a Korex Automatic Dishwasher Detergent to which 5% (w/w) sodium perborate monohydrate and 3% (w/w) TAED were added. Samples of these formulations were placed at room temperature (21-23°C) or at 38°C and 80% relative humidity for four weeks. Results are shown in Figs. 21 and 22.
  • Example 13 Oxidatively Sable Protease/ Oxidatively Stable Amylase Stability Studies
  • Enzyme granules containing either: 1) wild-type protease and wild- type amylase; or 2) bleach stable protease (GG36-M222S) made by the methods described in US Re 34606 and bleach stable amylase (AA20- M15T/W138Y/M197T) were dissolved in buffer containing 0.1 M sodium borate pH 10.2 and 0.005% Tween 80 at a concentration of 12.5 mg of each enzyme.
  • To 9 ml of these solutions was added either 1 ml distilled water or 1 ml 30% hydrogen peroxide. After incubation of the solutions at 25°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.
  • the data show that the combination of a bleach-stable amylase mutant and a bleach-stable protease mutant, both with mutations at amino acid residues sensitive to oxidation, provides the combined benefits of protease and amylase in a formulation resistant to inactivation by bleach.
  • the combination of a bleach-stable amylase and a bleach-stable protease retains most of its initial activity after 30 minutes in bleach, while the combination of wild-type enzymes loses over 80% of its initial activity in the same period of time.
  • 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.
  • 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 li cheniformis alpha amylase available from Novo Nordisk, Denmark,), 5.2 mg/ml Spezyme® AA20 ( Bacill us li cheniformis 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.
  • an amylase assay buffer 50 mM acetate buffer, pH 6.7, 5mM CaCl 2 , .002% Tween 20.
  • 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 Spezyme®AA20.
  • 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
  • Corn starch/india ink soiled cotton swatches PEPD 7435WRL obtained from Scientific Services S/D, Inc., Sparrow Bush, N.Y.
  • 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 (e.g., protease or lipase) .
  • Amylases used were the Termamyl® 60T amylase
  • 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
  • SA8® commercially available from Amway Corp., Ada, Michigan
  • Purex® commercially available from the Dial Corp., Phoenix, Arizona
  • Mutant amylase M15T/W138Y/M197T
  • 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 55*C.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID N0:2: TGGGACGCTG GCGCAGTACT TTGAATGGT 29
  • MOLECULE TYPE DNA (genomic)
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  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:19: CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
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  • GACTGTCCAA ACATGGTTTA AGCCGCTTGC TTACGCTTTT ATTCTCACAA GGGAATCTGG 1320
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:42: CACCTAATTA AAGCTTTCAC ACATTTTCAT TTT 33
  • MOLECULE TYPE DNA (genomic)
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  • MOLECULE TYPE DNA (genomic)
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Abstract

Laundry detergents comprising novel alpha-amylase mutants derived from the DNA sequences of naturally occuring or recombinant alpha-amylases are disclosed. The mutant alpha-amylases, in general, are obtained by in vitro modifications of a precursor DNA sequence encoding the naturally occuring or recombinant alpha-amylase to generate the substitution (replacement) or deletion of one or more oxidizable amino acid residues in the amino acid sequence of a precursor alpha-amylase. Such laundry detergents containing mutant alpha-amylases have increased cleaning ability stability and/or altered pH performance profiles and/or altered thermal stability as compared to the precursor.

Description

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 alpha- amylase, 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, -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. li cheniformis, B. amyloliquefaciens, B. subtilis, or B. stearothermophilus . In recent years the preferred enzymes in commercial use have been those from B. li cheniformis because of their heat stability and performance, at least at neutral and mildly alkaline pH's.
Previously there have been studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases (Vihinen, M. et al. (1990) J. 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. li cheniformis amylase, the rationale for making substitutions at histidine residues was that B. li cheniformis amylase (known to be thermostable) when compared to other similar Bacill us 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 <5.5). On the other hand, amylases may be used in commercial dish care or laundry detergents, which often contain oxidants such as bleach or peracids, and which are used in much more alkaline conditions.
In order to alter the stability or activity profile of amylase enzymes under varying conditions, it has been found that selective replacement, substitution or deletion of oxidizable amino acids, such as a methionine, tryptophan, tyrosine, histidine or cysteine, results in an altered profile of the variant enzyme as compared to its precursor. Because currently commercially available amylases are not acceptable (stable) under various conditions, there is a need for an amylase having an altered stability and/or activity profile. This altered stability (oxidative, thermal or pH performance profile) can be achieved while maintaining adequate enzymatic activity, as compared to the wild-type or precursor enzyme. The characteristic affected by introducing such mutations may be a change in oxidative stability while maintaining thermal stability or vice versa . Additionally, the substitution of different amino acids for an oxidizable amino 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 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 or in combination with the substitution of one or more methionine residue in the precursor alpha-amylase. Such mutant alpha-amylases, in general, are obtained by in vi tro modification of a precursor DNA sequence encoding a naturally occurring or recombinant alpha-amylase to encode the substitution or deletion of one or more amino acid residues in a precursor amino acid sequence.
Preferably the substitution or deletion of one or more amino acid in the amino acid sequence is due to the replacement or deletion of one or more methionine, tryptophan, cysteine, histidine or tyrosine residues in such sequence, most preferably the residue which is changed is a methionine residue. The oxidizable amino acid residues may be replaced by any of the other 20 naturally occurring amino acids. If the desired effect is to alter the oxidative stability of the precursor, the amino acid residue may be substituted with a non-oxidizable amino acid (such as alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, or valine) or another oxidizable amino acid (such as cysteine, methionine, tryptophan, tyrosine or histidine, listed in order of most easily oxidizable to less readily oxidizable) . Likewise, if the desired effect is to alter thermostability, any of the other 20 naturally occurring amino acids may be substituted (i.e., cysteine may be substituted for methionine) .
Preferred laundry detergents comprise mutants comprising the substitution of a methionine residue equivalent to any of the methionine residues found in B. li cheniformi s alpha-amylase (+8, +15, +197, +256, +304, +366 and +438) . Most preferably the methionine to be replaced is a methionine at a position equivalent to position +197 or +15 in B. li cheniformi s alpha-amylase. Preferred substitute amino acids to replace the methionine at position +197 are alanine ' (A) , isoleucine (I), threonine (T) or cysteine (C) . The preferred substitute amino acids at position +15 are leucine (L) , threonine (T) , asparagine (N) , aspartate (D) , serine (S) , valine (V) and isoleucine (I), although other substitute amino acids not specified above may be useful. Two specifically preferred mutants of the present invention are M197T and M15L.
Another embodiment of this invention relates to laundry detergents comprising mutants comprising the substitution of a tryptophan residue equivalent to any of the tryptophan residues found in B. licheniformis alpha-amylase (see Fig. 2). Preferably the tryptophan to be replaced is at a position equivalent to +138 in B. licheniformis alpha-amylase. A mutation (substitution) at a tryptophan residue may be made alone or in combination with mutations at other oxidizable amino acid residues. Specifically, it may be advantageous to modify by substitution at least one tryptophan in combination with at least one methionine (for example, the double mutant +138 +197) .
The alpha-amylase mutants 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 alpha- amylases according to the invention which has a pH above about 10 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. 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 alpha- amylase mutants derived from a Bacill us strain such as B . li cheniformis , B. amyloliquefaciens , and B. stearothermophilus, and most preferably from Bacillus li cheniformis .
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. li cheniformis . This novel form, designated as the A4 form, has an additional four alanine residues at the N- terminus of the secreted amylase. (Fig. 4b.) Derivatives or mutants of the A4 form of alpha-amylase are encompassed within the present invention. By derivatives or mutants of the A4 form, it is meant that the present invention comprises the A4 form alpha- amylase containing one or more additional mutations such as, for example, mutation (substitution, replacement or deletion) of one or more oxidizable amino acid(s).
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, cellulases, proteases, Upases or other amylase enzymes. Particularly preferred are laundry detergent compositions comprising a M15X/W138X/M197X mutant, and most preferably a M15T/W138Y/M197T ("TYT") 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. li cheniformis (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. li cheniformis (NCIB8061), Seq ID No 32.
Fig. 3a-3b shows an alignment of primary structures of Ba cill us alpha-amylases. The B. li cheniformis amylase (Am-Lich) , Seq ID No 33, is described by Gray, G. et al. (1986) J. Bact. 166:635-643; the B. amyloliquefaci ens amylase (Am-Amylo) , Seq ID No 34, is described by Takkinen, K. et al. (1983) J. Biol. Chem. 258:1007- 1013; and the B . stearothermophil us (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 alpha- amylase from B . li cheniformis NCIB8061, Seq ID No 37. Numbering is from the N-terminus, starting with the four additional alanines . Fig. 5 shows plasmid pA4BL wherein BLAA refers to B. li cheniformi s alpha-amylase gene, PstI to Sstl; AmpR refers to the ampicillin- resistant gene from pBR322; and CAT refers to the Chloramphenicol- resistant gene from pC194.
Fig. 6 shows the signal sequence-mature protein junctions for B. 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 (Spezyme® AA20 and M197L (A4 form) with 0.88M H202 at pH 5.0, 25°C.
Fig. 7b shows inactivation of certain alpha-amylases (Spezyme® AA20, M197T) with 0.88M H202 at pH 10.0, 25°C.
Fig. 7c shows inactivation of certain alpha-amylases (Spezyme® AA20, M15L) with 0.88M H202 at pH 5.0, 25°C.
Fig. 8 shows a schematic for the production of M197X cassette mutants.
Fig. 9 shows expression of M197X variants.
Fig. 10 shows thermal stability of M197X variants at pH 5.0, 5mM CaCl2 at 95°C for 5 mins.
Figs. 11a and lib show inactivation of certain amylases in automatic dish care detergents. Fig. 11a shows the stability of certain amylases in Cascade™ (a commercially available dish care product) at 65°C in the presence or absence of starch. Fig. lib shows the stability of certain amylases in Sunlight™ (a commercially available dish care product) at 65°C in the presence or absence of starch. Fig. 12 shows a schematic for the production of M15X cassette mutants.
Fig. 13 shows expression of M15X variants.
Fig. 14 shows specific activity of M15X variants on soluble starch.
Fig. 15 shows heat stability of M15X variants at 90°C, pH 5.0, 5mM CaCl2, 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. li cheniformis wild-type.
Fig. 17 shows the inactivation of B. li cheniformis alpha-amylase (AA20 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. licheniformi s alpha-amylase (AA20 at 0.22 mg/ml) with chloramine-T at pH 4.0 as compared to variants M197A (4.3 mg/ml) and M197L (0.53 mg/ml) .
Fig. 19 shows the reaction of B. li cheniformis alpha-amylase (AA20 at 0.75 mg/ml) with chloramine-T at pH 5.0 as compared to double variants M197T/ 138F (0.64 mg/ml) and M197T/W138Y (0.60 mg/ml) .
Fig. 20 shows the stability testing results of various alpha- amylase multiple mutants incorporated in automatic dish detergent (ADD) formulations at temperatures from room temperature increased to 65°C.
Fig. 21 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at room temperature over 0-30 days, as determined by percent activity remaining over time. Fig. 22 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at 38°C (100°F) with 80% relative humidity over 0-30 days.
Fig. 23 shows the pH activity profile of certain amylases on a Phadebas substrate at 25βC at neutral and alkaline pH.
Fig. 24 shows the stability of certain amylases to peracetic acid over time at pH 9.3 and 52βC.
Fig. 25 shows the relative cleaning ability of amylase according to the invention ("TYT") compared to Termamyl amylase in liquid laundry detergent at 40βC in terms of reflectance (delta from control) vs. ppm amylase added.
Fig. 26 shows the relative cleaning ability of amylase according to the invention ("TYT") compared to Termamyl amylase in liquid laundry detergent at 55βC 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 applications 07/785,624 and 07/785,623 and US Patent 5,180,669, issued January 19, 1993, incorporated herein by reference) . Furthermore, use of an amylase in the presence of oxidants, such as in bleach- or peracid- containing detergents, may result in partial or complete inactivation of the amylase. Therefore, the present invention focuses on altering the oxidative sensitivity of amylases which are added to laundry detergents. The mutant enzymes in the laundry detergents of the present invention may also have an altered pH profile and/or altered thermal stability which may be due to the enhanced oxidative stability of the enzyme at low or high pH's.
Alpha-amylase as used herein includes naturally occurring amylases as well as recombinant amylases. Preferred amylases in the present invention are alpha-amylases derived from B. li cheniformis or B. stearothermophil us, including the A4 form of alpha-amylase derived from B. li cheniformis as described herein, as well as fungal alpha- amylases such as those derived from Aspergillus (i.e., A. oryzae and A. ni ger) .
Recombinant alpha-amylases refers to an alpha-amylase in which the DNA sequence encoding the naturally occurring alpha-amylase is modified to produce a mutant DNA sequence which encodes the substitution, insertion or deletion of one or more amino acids in the alpha-amylase sequence. Suitable modification methods are disclosed herein, and also in commonly owned US Patents 4,760,025 and 5,185,258, the disclosure of which are incorporated herein by reference.
Homologies have been found between almost all endo-amylases sequenced to date, ranging from plants, mammals, and bacteria (Nakajima, R.T. et al. (1986) Appl. Microbiol. Biotechnol. 23:355- 360; Rogers, J.C. (1985) Biochem. Biophys . Res. Commun. 128:470- 476) . There are four areas of particularly high homology in certain Baci llus 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 Bacill us endo-amylases (Feng, D.F. and Doolittle, R.F. (1987) J. Molec. Evol. 35:351-360) . The relative sequence homology between B. stearothermophil us and B. li cheniformi s amylase is about 66%, as determined by Holm, L. et al. (1990) Protein Engineering 3_ (3) pp. 181-191. The sequence homology between B . li cheniformi s and B . amyloliquefaci ens 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 amylases or other enzymes. For example, structural homology between fungal amylases and bacterial (Bacill us) 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 li cheniformi s alpha-amylase are identified herein for substitution or deletion, as are all methionine, histidine, tryptophan, cysteine and tyrosine positions. The amino acid position number (i.e., +197) refers to the number assigned to the mature Bacill us li cheniformi s alpha-amylase sequence presented in Fig. 2. The invention, however, is not limited to the mutation of this particular mature alpha-amylase [ B. li cheniformis ) but extends to precursor alpha-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in B. li cheniformis alpha-amylase. A residue (amino acid) of a precursor alpha-amylase is equivalent to a residue of B. li cheniformi s alpha-amylase if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B . li cheniformi s alpha-amylase (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally) .
In order to establish homology to primary structure, the amino acid sequence of a precursor alpha-amylase is directly compared to the B. li cheniformis 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. ni ger (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 Taka- amylase A (Holm, L. et al. (1990) Protein Engineering 3:181-191) . The four highly conserved regions shown in Fig. 3 contain many residues thought to be part of the active-site (Matsuura, Y. et al. (1984) J. Biochem. (Tokyo) 95:697-702; Buisson, G. et al. (1987) EMBO J. 6:3909-3916; Vihinen, M. et al. (1990) J. Biochem. 107:267- 272) including, in the li cheniformis 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 . subti lis aprE promoter. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include 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 Bacill us strain. Preferably an alpha-amylase negative Bacill us strain (genes deleted) and/or an alpha-amylase and protease deleted Bacill us strain such as Bacillus subtilis strain BG2473
(AamyE, apr, 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 between about 5.0 and about 10.0 and most preferably between about 6.0 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 ("TYT") .
Another embodiment of the present invention comprises the combination of the mutant alpha-amylase enzymes described herein in combination with other enzymes (i.e., proteases, lipases, cellulases, etc.), and preferably oxidatively stable proteases. Suitable oxidatively stable proteases include genetically engineered proteases such as those described in US Re 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. amyloliquefaci ens, are described in US Re 34606. Suitable methods for determining "equivalent" positions in other subtilisins are provided in Re 34606, EP 257,446 and USSN 212,291, which are incorporated herein by reference.
Abbreviations used herein, particularly three letter or one letter notations for amino acids are described in Dale, J.W., Molecular Genetics of Bacteria, John Wiley & Sons, (1989) Appendix B.
Experimental
Example 1 Substitutions for the Methionine Residues in B . li cheniformis Alpha-Amylase The alpha-amylase gene (Fig. 1) was cloned from B. li cheniformi s NCIB8061 obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland (Gray, G. et al. (1986) J. Bacteriology 166:635-643) . The 1.72kb Pstl-Sstl fragment, encoding the last three residues of the signal sequence; the entire mature protein and the terminator region was subcloned into M13MP18. A synthetic terminator was added between the Bell and Sstl sites using a synthetic oligonucleotide cassette of the form:
Bell Sstl
5 ' GATCAAAACATAAAAAACCGGCCTTGGCCCCGCCGGTTTTTTATTATTTTTGAGCT 3 ' 3' TTTTGTATTTTTTGGCCGGAACCGGGGCGGCCAAAAAATAATAAAAAC 5 '
Seq ID No 1
designed to contain the B. amyloliquefaci ens subtilisin transcriptional terminator (Wells et al. (1983) Nucleic Acid Research 11:7911-7925) .
Site-directed mutagenesis by oligonucleotides used essentially the protocol of Zoller, M. et al . (1983) Meth. Enzymol. 100:468-500: briefly, 5 ' -phosphorylated oligonucleotide primers were used to introduce the desired mutations on the M13 single-stranded DNA template using the oligonucleotides listed in Table I to substitute for each of the seven methionines found in B . li cheniformis alpha- amylase. 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. li cheniformis Alpha-Amylase
M8A 5'-T GGG ACG CTG GCG CAG TAC TTT GAA TGG T-3' Seq ID No 2
Scal+
M15L 5'-TG ATG CAG TAC TTT GAA TGG TAC CTG CCC AAT GA-3 ' Seq ID No 3 Scal+ Kpnl+
M197L 5»-GAT TAT TTG TTG TAT GCC GAT ATC GAC TAT GAC CAT-3 » Seq ID No 4
EcoRV+
M256A 5'-CG GGG AAG GAG GCC TTT ACG GTA GCT-3' Seq ID No 5
Stul+
M304L 5 '-GC GGC TAT GAC TTA AGG AAA TTG C-3' Seq ID No 6
AfIII+
M366A 5'-C TAC GGG GAT GCA TAC GGG ACG A-3 ' Seq ID No 7
Nsil+
M366Y 5'-C TAC GGG GAT TAC TAC GGG ACC AAG GGA GAC TCC C-3' Seq ID No 8
Styl+
M438A 5'-CC GGT GGG GCC AAG CGG GCC TAT GTT GGC CGG CAA A-3' Seq ID No 9
Sfil+
Bold letter indicate base changes introduced by oligonucleotide.
Codon changes indicated in the form M8A, where methionine (M) at position +8 has been changed to alanine (A) . Underlining indicates restriction endonuclease site introduced by oligonucleotide.
The heteroduplex was used to transfect E. coli mutL cells (Kramer et al. (1984) Cell 38:879) and, after plaque-purification, clones were analyzed by restriction analysis of the RF1 's. Positives were confirmed by dideoxy sequencing (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and the Pstl-Sstl fragments for each subcloned into an E. coli vector, plasmid pA4BL.
Plasmid pA4BL
Following the methods described in US application 860,468 (Power et al.), which is incorporated herein by reference, a silent Pstl site was introduced at codon +1 (the first amino-acid following the signal cleavage site) of the aprE gene from pS168-l (Stahl, M.L. and Ferrari, E. (1984) J. Bacter. 158:411-418) . The aprE promoter and signal peptide region was then cloned out of a pJHIOl plasmid (Ferrari, F.A. et al. (1983) J. Bacter. 154:1513-1515) as a Hindlll-Pstl fragment and subcloned into the pUC18-derived plasmid JM102 (Ferrari, E. and Hoch, J.A. (1989) Bacillus, ed. C.R. Harwood, Plenum Pub., pp. 57-72) . Addition of the Pstl-Sstl fragment from B. li cheniformis alpha-amylase gave pA4BL (Fig. 5) 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. subtili s 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 subtili s strain BG2473 was a derivative of 1168 which had been deleted for amylase (Δa-τjyE) and two proteases (Aapr, Anpr) (Stahl, M.L. and Ferrari, E., 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) 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 li cheniformis amylase and the variant M197T were made from a very similar construction (see Fig. 6) . Specifically, the 5' end of the A4 construction was subcloned on an EcoRI-Sstll fragment, from pA4BL (Fig. 5) into M13BM20 (Boehringer Mannheim) in order to obtain a coding-strand template for the mutagenic oligonucleotide below:
5 '-CAT CAG CGT CCC ATT AAG ATT TGC AGC CTG CGC AGA CAT GTT GCT-3 '
Seq ID No 10
This primer eliminated the codons for the extra four N-terminal alanines, correct forms being screened for by the absence of the Pstl site. Subcloning the EcoRI-Sstll fragment back into the pA4BL vector (Fig. 5) gave plasmid pBLapr. The M197T substitution could then be moved, on a Sstll-Sstl fragment, out of pA4BL (M197T) into the complementary pBLapr vector to give plasmid pBLapr (M197T) . N- terminal analysis of the amylase expressed from pBLapr in B . subtilis showed it to be processed with the same N-terminus found in B. li cheniformi s alpha-amylase.
Example 2 Oxidative Sensitivity of Methionine Variants B. li cheniformi s alpha-amylase, such as Spezyme® AA20 (commercially available from Genencor International, Inc.), is inactivated rapidly in the presence of hydrogen peroxide (Fig. 7) . Various methionine variants were expressed in shake-flask cultures of B. subtilis and the crude supernatants purified by ammonium sulphate cuts. The amylase was precipitated from a 20% saturated ammonium sulphate supernatant by raising the ammonium sulphate to 70% saturated, and then resuspended. The variants were then exposed to
0.88M hydrogen peroxide at pH 5.0, at 25°C. Variants at six of the methionine positions in B. li cheniformis alpha-amylase were still subject to oxidation by peroxide while the substitution at position +197 (M197L) showed resistance to peroxide oxidation. (See Fig. 7.) However, subsequent analysis described in further detail below showed that while a variant may be susceptible to oxidation at pH 5.0, 25°C, it may exhibit altered/enhanced properties under different conditions (i.e., liquefaction) .
Example 3 Construction of All Possible Variants at Position 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 5' -GAT TAT TTG GCG TAT GCC GAT ATC GAC TAT GAC CAT-3'
EcoRV+
Clal- Seq ID No 11
which also inserted an EcoRV site (codons 200-201) to replace the Clal site (codons 201-202).
2) Then primer LAAM12 (Table II) was used to introduce another silent restriction site (BstBI) over codons 186-188.
3) The resultant M197A (BstBI+, EcoRV+) variant was then subcloned (Pstl-Sstl fragment) into plasmid pA4BL and the resultant plasmid digested with BstBI and EcoRV and the large vector-containing fragment isolated by electroelution from agarose gel. 4) Synthetic primers LAAM14-30 (Table II) were each annealed with the largely complementary common primer LAAM13 (Table II). The resulting cassettes encoded for all the remaining naturally occurring amino acids at position +197 and were ligated, individually, into the vector fragment prepared above.
TABLE II
Synthetic Oligonucleotides Used for Cassette Mutagenesis to Produce M197X Variants
IAAM12 GG GAA GTT TCG AAT GAA AAC G Seq ID No 12
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-)
LAAM20 Y197 Seq ID No 20 (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 G197 Seq ID No 22 c (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG GGC TAT GCC GAC (EcoRV-
CD
(/.
LAAM23 Q197 Seq ID No 23
H (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG CAA TAT GCC GAC (EcoRV- C Hm LAAM24 N197 Seq ID No 24
(BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG AAC TAT GCC GAC (EcoRV-
IAAM27 E197 Seq ID No 27 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG GAA TAT GCC GAC (EcoRV-
LAAM28 C197 Seq ID No 28 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG TGT TAT GCC GAC (EcoRV-
LAAM29 W197 Seq ID No 29 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG TGG TAT GCC GAC (EcoRV-
LAAM30 R197 Seq ID No 30 (BstBI) CG AAT GAA AAC GGC AAC TAT GAT TAT TTG AGA TAT GCC GAC (EcoRV-
The cassettes were designed to destroy the EcoRV site upon ligation, thus plasmids from E. coli transformants were screened for loss of this unique site. In addition, the common bottom strand of the cassette contained a frame-shift and encoded a Nsil site, thus transformants derived from this strand could be eliminated by screening for the presence of the unique Nsil site and would not be expected, in any case, to lead to expression of active amylase.
Positives by restriction analysis were confirmed by sequencing and transformed in B. subtilis for expression in shake-flask cultures (Fig. 9) . The specific activity of certain of the M197X mutants was then determined using a soluble substrate assay. The data generated using the following assay methods are presented below in Table III.
Soluble Substrate Assay: A rate assay was developed based on an end-point assay kit supplied by Megazyme (Aust.) Pty. Ltd.: Each vial of substrate (p-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10ml of sterile water, followed by a 1 to 4 dilution in assay buffer (50mM maleate buffer, pH 6.7, 5mM calcium chloride, 0.002% Tween20). Assays were performed by adding lOμl of amylase to 790μl of the substrate in a cuvette at 25°C. Rates of hydrolysis were measured as the rate of change of absorbance at 410nm, after a delay of 75 seconds. The assay was linear up to rates of 0.4 absorption units/min.
The amylase protein concentration was measured using the standard Bio-Rad assay (Bio-Rad Laboratories) based on the method of Bradford, M. (1976) Anal. Biochem. 72:248) using bovine serum albumin standards. Starch Hydrolysis Assay: The standard method for assaying the alpha-amylase activity of Spezyme® AA20 was used. This method is described in detail in Example 1 of USSN 07/785,624, incorporated herein by reference. Native starch forms a blue color with iodine but fails to do so when it is hydrolyzed into shorter dextrin molecules. The substrate is soluble Lintner starch 5gm/liter in phosphate buffer, pH 6.2 (42.5gm/liter potassium dihydrogen phosphate, 3.16gm/liter sodium hydroxide) . The sample is added in 25mM calcium chloride and activity is measured as the time taken to give a negative iodine test upon incubation at 30°C. Activity is recorded in liquefons per gram or ml (LU) calculated according to the formula:
LU/ml or LU/g = 570 x D
V x t
Where LU=liquefon unit
V=volume of sample (5ml) t=dextrinization time (minutes)
D=dilution factor=dilution volume/ml or g of added enzyme.
TABLE III
SPECIFIC ACTIVITY (as % of AA20 value) on
ALPHA-AMYLASE Soluble Substrate Starch
Spezyme® AA20 100 100
A4 form 105 115
M15L (A4 form) 93 94
M15L 85 103
M197T (A4 form) 75 83
M197T 62 81
M197A (A4 form) 88 89
M197C 85 85
M197L (A4 form) 51 17
Example 4 Characterization of Variant M15L Variant M15L made as per the prior examples did not show increased amylase activity (Table III) and was still inactivated by hydrogen peroxide (Fig. 7) . It did, however, show significantly increased performance in jet-liquefaction of starch, especially at low pH as shown in Table IV below.
Starch liquefaction was typically performed using a Hydroheater M 103-M steam jet equipped with a 2.5 liter delay coil behind the mixing chamber and a terminal back pressure valve. Starch was fed to the jet by a Moyno pump and steam was supplied by a 150 psi steam line, reduced to 90-100 psi. Temperature probes were installed just after the Hydroheater jet and just before the back pressure valve.
Starch slurry was obtained from a corn wet miller and used within two days. The starch was diluted to the desired solids level with deionized water and the pH of the starch was adjusted with 2% NaOH or saturated Na2C03. 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°-107°C. Samples of starch were transferred from the jet cooker to a 95°C second stage liquefaction and held for 90 minutes .
The degree of starch liquefaction was measured immediately after the second stage liquefaction by determining the dextrose equivalence (DE) of the sample and by testing for the presence of raw starch, both according to the methods described in the Standard Analytical Methods of the Member Companies of the Corn Refiners Association, Inc., sixth edition. Starch, when treated generally under the conditions given above and at pH 6.0, will yield a liquefied starch with a DE of about 10 and with no raw starch. Results of starch liquefaction tests using mutants of the present invention are provided in Table IV. TABLE IV
Performance of Variants M15L (A4 form) and M15L in Starch Liquefaction pH DE after 90 Mins .
Spezyme® AA20 5.9 9.9
M15L (A4 form) 5.9 10.4
Spezyme® AA20 5.2 1.2
M15L (A4 form) 5.2 2.2
Spezyme® AA20 5.9 9.3*
M15L 5.9 11.3*
Spezyme® AA20 5.5 3.25**
M15L 5.5 6.7**
Spezyme® AA20 5.2 0.7**
M15L . 5.2 3.65**
* average of three experiments ** average of two experiments
Example 5 Construction of M15X Variants Following generally the processes described in Example 3 above, all variants at M15 (M15X) were produced in native B. li cheniformi s by cassette mutagenesis, as outlined in Fig. 12:
1) Site directed mutagenesis (via primer extension in M13) was used to introduce unique restriction sites flanking the M15 codon to facilitate insertion of a mutagenesis cassette. Specifically, a BstBI site at codons 11-13 and a Mscl site at codons 18-20 were introduced using the two oligonucleotides shown below:
M15XBstBl 5'-G ATG CAG TAT TTC GAA CTGG TAT A-3'
BstBI Seq ID No 48
M15XMSC1 5'-TG CCC AAT GAT GGC CAA CAT TGG AAG-3 '
Mscl Seq ID No 49
2) The vector for M15X cassette mutagenesis was then constructed by subcloning the Sfil-Sstll fragment from the mutagenized amylase
(BstBl+, Mscl+) into plasmid pBLapr. The resulting plasmid was then digested with BstBI and Mscl and the large vector fragment isolated by electroelution from a polyacrylamide gel.
3) Mutagenesis cassettes were created as with the M197X variants. Synthetic oligomers, each encoding a substitution at codon 15, were annealed to a common bottom primer. Upon proper ligation of the cassette to the vector, the Mscl is destroyed allowing for screening of positive transformants by loss of this site. The bottom primer contains an unique SnaBl site allowing for the transformants derived from the bottom strand to be eliminated by screening for the SnaBl site. This primer also contains a frameshift which would also eliminate amylase expression for the mutants derived from the common bottom strand.
The synthetic cassettes are listed in Table V and the general cassette mutagenesis strategy is illustrated in Figure 12.
TABLE V
Synthetic Oligonucleotides Used for Cassette Mutagenesis to Produce M15X Variants
M15A (BstBI > c GAA TGG TAT GCT CCC AAT GAC GG (Mscl) Seq ID No 50
M15R (BstBI C GAA TGG TAT CGC CCC AAT GAC GG (Mscl) Seq ID No 51
M15N (BstBI C GAA TGG TAT AAT CCC AAT GAC GG (Mscl) Seq ID No 52
M15D (BstBI C GAA TGG TAT GAT CCC AAT GAC GG (Mscl) Seq ID No 53
M15H (BstBI C GAA TGG TAT CAC CCC AAT GAC GG (Mscl) Seq ID No 54
M15K BstBI C GAA TGG TAT AAA CCC AAT GAC GG (Mscl) Seq ID No 55
M15P BstBI C GAA TGG TAT CCG CCC AAT GAC GG (Mscl) Seq ID No 56
M15S BstBI C GAA TGG TAT TCT CCC AAT GAC GG (Mscl) Seq ID No 57
M15T BstBI; C GAA TGG TAC ACT CCC AAT GAC GG (Mscl) Seq ID No 58
M15V BstBI C GAA TGG TAT GTT CCC AAT GAC GG (Mscl) Seq ID No 59
M15C BstBI C GAA TGG TAT TGT CCC AAT GAC GG (Mscl) Seq ID No 60
M15Q BstBI C GAA TGG TAT CAA CCC AAT GAC. GG (Mscl) Seq ID No 61
M15E BstBI C GAA TGG TAT GAA CCC AAT GAC GG (Mscl) Seq ID No 62
M15G BstBI) C GAA TGG TAT GGT CCC AAT GAC GG (Mscl) Seq ID No 63
M15I BstBI) C GAA TGG TAT ATT CCC AAT GAC GG (Mscl) Seq ID No 64
M15F BstBI) c GAA TGG TAT TTT CCC AAT GAC GG (Mscl) Seq ID No 65
M15W BstBI) c GAA TGG TAC TGG CCC AAT GAC GG (Mscl) Seq ID No 66
M15Y BstBI) c GAA TGG TAT TAT CCC AAT GAC GG (Mscl) Seq ID No 67
M15X ( Mscl) cc GTC ATT GGG ACT ACG TAC CAT T (BstBI) Seq ID No 68
(bottom strand)
Underline indicates codon changes at amino acid position 15.
Conservative substitutions were made in some cases to prevent introduction of new restriction sites. Example 6 Bench Liquefaction with M15X Variants Eleven alpha-amylase variants with substitutions for M15 made as per Example 5 were assayed for activity, as compared to Spezyme® AA20 (commercially available from Genencor International, Inc.) in liquefaction at pH 5.5 using a bench liquefaction system. The bench scale liquefaction system consisted of a stainless steel coil
(0.25 inch diameter, approximately 350 ml volume) equipped with a 7 inch long static mixing element approximately 12 inches from the anterior end and a 30 psi back pressure valve at the posterior end.
The coil, except for each end, was immersed in a glycerol-water bath equipped with thermostatically controlled heating elements that maintained the bath at 105-106°C.
Starch slurry containing enzyme, maintained in suspension by stirring, was introduced into the reaction coil by a piston driven metering pump at about 70 ml/min. The starch was recovered from the end of the coil and was transferred to the secondary hold (95°C for 90 minutes) . Immediately after the secondary hold, the DE of the liquefied starch was determined, as described in Example . The results are shown in Fig. 16.
Example 7 Characterization of M197X Variants As can be seen in Fig. 9, there was a wide range of amylase activity (measured in the soluble substrate assay) expressed by the M197X (A4 form) variants. The amylases were partially purified from the supernatants by precipitation with two volumes of ethanol and resuspension. They were then screened for thermal stability
(Fig. 10) by heating at 95°C for 5 minutes in lOmM acetate buffer pH 5.0, in the presence of 5mM calcium chloride; the A4 wild-type retained 28% of its activity after incubation. For M197W and M197P we were unable to recover active protein from the supernatants. Upon sequencing, the M197H variant was found to contain a second mutation, N190K. M197L was examined in a separate experiment and was one of the lowest thermally stable variants. There appears to be a broad correlation between expression of amylase activity and thermal stability. The li cheniformis amylase is restricted in what residues it can accommodate at position 197 in terms of retaining or enhancing thermal stability: cysteine and threonine are preferred for maximal thermal stability under these conditions whereas alanine and isoleucine are of intermediate stability. However, other substitutions at position +197 result in lowered thermal stability which may be useful for other applications. Additionally, different substitutions at +197 may have other beneficial properties, such as altered pH performance profile or altered oxidative stability. For example, the M197C variant was found to inactivate readily by air oxidation but had enhanced thermal stability. Conversely, compared to the M197L variant, both M197T and M197A retained not only high thermal stability (Fig. 10), but also high activity (Table III), while maintaining resistance to inactivation by peroxide at pH 5 to pH 10 (Fig. 7) .
Example 8 Stability and Performance in Detergent Formulation The stability of the M197T (A4 form), M197T and M197A (A4 form) was measured in automatic dish care detergent (ADD) matrices. 2ppm
Savinase™ (a protease, commercially available from Novo Industries, of the type commonly used in ADD) were added to two commercially available bleach-containing ADD's: Cascade™ (Procter and Gamble, Ltd.) and Sunlight™ (Unilever) and the time course of inactivation of the amylase variants and Termamyl™ (a thermally stable alpha-amylase available from Novo Nordisk, A/S) followed at 65°C. The concentration of ADD product used in both cases was equivalent to 'pre-soak' conditions: 14gm product per liter of water (7 grams per gallon hardness) . As can be seen (Figs. 11a and lib) , both forms of the M197T variant were much more stable than
Termamyl™ and M197A (A4 form) , which were inactivated before the first assay could be performed. This stability benefit was seen in the presence or absence of starch as determined by the following protocol. Amylases were added to 5ml of ADD and Savinase™, prewarmed in a test tube and, after vortexing, activities were assayed as a function of time, using the soluble substrate assay. The "+ starch" tube had spaghetti starch baked onto the sides (140°C, 60 mins.). The results are shown in Figs. 11a and lib.
Example 9 Characterization of M15X Variants All M15X variants were propagated in Bacillus 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 M15X amylases have specific activities greater than that of Spezyme® AA20. A benchtop heat stability assay was performed on the variants by heating the amylase at 90°C for 5 min. in 50 mM acetate buffer pH 5 in the presence of 5 mM CaCl2 (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 60% 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 pH- liquefaction performance and/or increased specific activity.
Example 10 Tryptophan Sensitivity to Oxidation Chloramine-T (sodium N-chloro-p-toluenesulfonimide) is a selective oxidant, which oxidizes methionine to methionine sulfoxide at neutral or alkaline pH. At acidic pH, chloramine-T will modify both methionine and tryptophan (Schechter, Y., Burstein, Y. and Patchornik, A. (1975) Biochemistry 14 (20) 4497-4503) . Fig. 17 shows the inactivation of B. li cheniformis alpha-amylase with chloramine-T at pH 8.0 (AA20 = 0.65 mg/ml, M197A = 1.7 mg/ml, M197L = 1.7 mg/ml) . The data shows that by changing the methionine at position 197 to leucine or alanine, the inactivation of alpha- amylase can be prevented. Conversely, as shown in Fig. 18, at pH 4.0 inactivation of the M197A and M197L proceeds, but require more equivalents of chloramine-T (Fig. 18; AA20 = 0.22 mg/ml, M197A = 4.3 mg/ml, M197L = 0.53 mg/ml; 200 mM NaAcetate at pH 4.0) . This suggests that a tryptophan residue is also implicated in the chloramine-T mediated inactivation event. Furthermore, tryptic mapping and subsequent amino acid sequencing indicated that the tryptophan at position 138 was oxidized by chloramine-T (data not shown) . To prove this, site-directed mutants were made at tryptophan 138 as provided below:
Preparation of Alpha-Amylase Double Mutants W138 and Ml97 Certain variants of W138 (F, Y and A) were made as double mutants, with M197T (made as per the disclosure of Example 3) . The double mutants were made following the methods described in Examples 1 and 3. Generally, single negative strands of DNA were prepared from an M13MP18 clone of the 1.72kb coding sequence (Pst I-Sst I) of the B. li cheniformis 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 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 Sstll/Sstl fragment containing both W138X and M197T mutations was moved from M13MP18 into the expression vector pBLapr resulting in pBLapr (W138F, M197T) and pBLapr (W138Y, M197T) . The fragment containing unique BspE I and Xma I sites was cloned into pBLapr (BspE I, Xma I, M197T) since it is useful for cloning cassettes containing other amino acid substitutions at position 138.
Single Mutations at Amino Acid Position 138
Following the general methods described in the prior examples, certain single variants of W138 (F, Y, L, H and C) were made.
The 1.24kb Asp718-Sstl fragment containing the M197T mutation in plasmid pBLapr (W138X, M197T) of Example 7 was replaced by the wild-type fragment with methionine at 197, resulting in pBLapr (W138F), pBLapr (W138Y) and pBLapr (BspE I, Xma I) .
The mutants 138L, W138H and 138C were made by ligating synthetic cassettes into the pBLapr (BspE I, Xma I) vector using the following primers:
Tryptophan 138 to Leucine
CC GGA GAA CAC CTA ATT AAA GCC CTA ACA CAT TTT CAT TTT C
Seq ID No 45 Tryptophan 138 to Histidine
CC GGA GAA CAC CTA ATT AAA GCC CAC ACA CAT TTT CAT TTT C
Seq ID No 46 Tryptophan 138 to 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 5.0) . The results shown in Fig. 19 show that mutagenesis of tryptophan 138 has caused the variant to be more resistant to chloramine-T.
Example 11 Preparation of Multiple Mutants
Following the methods of Examples 1, 3, 5 and 10, the following multiple mutants were made: M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T and M15T/W138Y/M197T. Certain of these multiple mutants were previously exemplified, for example, W138Y/M197T was made and tested in Example 10. The multiple mutants were identified by restriction analysis.
Various multiple mutants within the scope of the present invention were further tested for performance as cleaning products (automatic dish care detergent) additives. These tests are detailed below.
Stability Testing
A 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg. Certain amylase mutants described above were added to this ADD solution to yield a rate of 0.4 when assayed by the Ceralpha method (Megazyme (Austr.) Pty. Ltd., Parramatta, NSW, Australia) . One set of samples was held at room temperature (21- 23°C) for about 30 min. (non-heated) . A second set of samples was warmed from room temperature to about 65°C after addition of the enzyme (heated) . 30 min. after addition of the enzyme, the activity of the amylase mutants was measured and the activity relative to the activity at the time of addition of the enzyme was calculated (relative activity %).
The results shown in Fig. 20 indicate that the methionine at position +197 of B . li cheniformis alpha-amylase should be modified for stability in a formulation comprising ADD + perborate + TAED.
Starch Hydrolysis Assay
A 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg and three cooked pieces of elbow macaroni were added. The amylase mutants described above were added to this ADD solution to yield a final concentration of 5 ppm active enzyme. The tubes were incubated at 50°C for about 30 min. and the concentration of reducing sugars released was measured against a glucose standard curve using the dinitrosalicylic acid method. Results are shown in Table VI.
Table VI
Reducing Sugar
Enzyme Concentration (g/1) 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°C. Dishes were loaded in an ASKO Model 770 dishwasher and washed at 45°C on the Quick Wash cycle using 10 g of automatic dishwashing detergent containing 5% perborate, 3% TAED and 11 mg of certain amylase enzyme(s) . The plates were weighed before soiling, after soiling and after washing, and the average % soil removed from all plates was calculated. The data are shown below in Table VII.
Table VII
The data show that the mutant enzymes provided a benefit greater than that provided by the wild-type. Wild-type amylase provided a 20% greater cleaning benefit in removing oatmeal than did ADD without amylase.
Example 12 Dish Care Cleaning Composition 1% (w/w) granules of wild-type and mutant amylases were formulated with a Korex Automatic Dishwasher Detergent to which 5% (w/w) sodium perborate monohydrate and 3% (w/w) TAED were added. Samples of these formulations were placed at room temperature (21-23°C) or at 38°C and 80% relative humidity for four weeks. Results are shown in Figs. 21 and 22.
The data show that the wild-type amylase activity, as measured by the Ceralpha method, decreased with increasing storage time in detergent. At room temperature, the mutant enzymes were completely stable. At 38°C and 80% relative humidity, all mutants were more stable than the wild-type.
The advantage of formulating an automatic dishwashing detergent with these mutant amylases is that these mutants are significantly more stable than the wild-type in the presence of perborate and TAED and they provide a significant performance benefit in removing starchy food stains in the wash.
Example 13 Oxidatively Sable Protease/ Oxidatively Stable Amylase Stability Studies Enzyme granules containing either: 1) wild-type protease and wild- type amylase; or 2) bleach stable protease (GG36-M222S) made by the methods described in US Re 34606 and bleach stable amylase (AA20- M15T/W138Y/M197T) were dissolved in buffer containing 0.1 M sodium borate pH 10.2 and 0.005% Tween 80 at a concentration of 12.5 mg of each enzyme. To 9 ml of these solutions was added either 1 ml distilled water or 1 ml 30% hydrogen peroxide. After incubation of the solutions at 25°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 Af er 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 75
The data show that the combination of a bleach-stable amylase mutant and a bleach-stable protease mutant, both with mutations at amino acid residues sensitive to oxidation, provides the combined benefits of protease and amylase in a formulation resistant to inactivation by bleach. The combination of a bleach-stable amylase and a bleach-stable protease retains most of its initial activity after 30 minutes in bleach, while the combination of wild-type enzymes loses over 80% of its initial activity in the same period of time.
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 li cheniformis alpha amylase available from Novo Nordisk, Denmark,), 5.2 mg/ml Spezyme® AA20 ( Bacill us li cheniformis 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 CaCl2, .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 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 Spezyme®AA20.
Example 15 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-n- cyclohexylaminoethane-sulfonic acid) and 0.005M calcium chloride at a pH of 10.0 at 25°C. The sample was mixed by vortexing and a 10 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 52βC as measured inside of the reaction tube. The caps of the tubes were sealed during the reaction. The pH of the buffer at 52βC 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 (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 90βC for one hour to inactivate any enzymes present (e.g., protease or lipase) . Amylases used were the Termamyl® 60T amylase
(wild type Bacillus li cheniformis amylase available from Novo Nordisk, Denmark) and the inventive amylase, M15T/W138Y/M197 .
1.0 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, N.J.) The wash liquor comprised distilled water with 10 ml of hard water concentrate added to result in a final concentration of 100 ppm Ca2+ ion and 50 ppm Mg2+. 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 15 minutes of agitation at lOOrpm, 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 40βC and 55βC. 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 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 55*C. Purex® was added to the wash liquor in a quantity of 1.0 gram/liter to bring the wash solution to a pH of 9.0 and a temperature of 40βC. 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.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(l) APPLICANT: GENENCOR INTERNATIONAL, INC.
(n) TITLE OF INVENTION: An Improved Laundry Detergent Composition Comprising Amylase
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(A) NAME: Stone, Christopher L.
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(2) INFORMATION FOR SEQ ID NO:5:
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(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCGGTGGGGC CAAGCGGGCC TATGTTGGCC GGCAAA 36
(2) INFORMATION FOR SEQ ID NO:10:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: CATCAGCGTC CCATTAAGAT TTGCAGCCTG CGCAGACATG TTGCT 45
(2) INFORMATION FOR SEQ ID NO: 11: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GATTATTTGG CGTATGCCGA TATCGACTAT GACCAT 36
(2) INFORMATION FOR SEQ ID NO: 12:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAAGTTTC GAATGAAAAC G 21
(2) INFORMATION FOR SEQ ID NO: 13:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTCGGCATAT GCATATAATC ATAGTTGCCG TTTTCATT 38
(2) INFORMATION FOR SEQ ID NO: 14:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:14: CGAATGAAAA CGGCAACTAT GATTATTTGA TCTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO: 15:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CGAATGAAAA CGGCAACTAT GATTATTTGT TCTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:16:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CGAATGAAAA CGGCAACTAT GATTATTTGG TTTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO: 17:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: DNA (genomic)
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CGAATGAAAA CGGCAACTAT GATTATTTGA GCTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO: 18:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGAATGAAAA CGGCAACTAT GATTATTTGC CTTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO: 19:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CGAATGAAAA CGGCAACTAT GATTATTTGA CATATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CGAATGAAAA CGGCAACTAT GATTATTTGT ACTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGAATGAAAA CGGCAACTAT GATTATTTGC ACTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CGAATGAAAA CGGCAACTAT GATTATTTGG GCTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CGAATGAAAA CGGCAACTAT GATTATTTGC AATATGCCGA C 41 (2) INFORMATION FOR SEQ ID NO:24:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CGAATGAAAA CGGCAACTAT GATTATTTGA ACTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:25:
(l) SEQUENCE CHARACTERISTIC'S:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GCAATGAAAA CGGCAACTAT GATTATTTGA AATATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:26:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CGAATGAAAA CGGCAACTAT GATTATTTGG ATTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:27:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CGAATGAAAA CGGCAACTAT GATTATTTGG AATATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:28: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CGAATGAAAA CGGCAACTAT GATTATTTGT GTATTGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:29:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CGAATGAAAA CGGCAACTAT GATTATTTGT GGTATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:30:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: CGAATGAAAA CGGCAACTAT GATTATTTGA GATATGCCGA C 41
(2) INFORMATION FOR SEQ ID NO:31:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1968 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
AGCTTGAAGA AGTGAAGAAG CAGAGAGGCT ATTGAATAAA TGAGTAGAAA GCGCCATATC 60
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 TGACGGCCAA CATTGGAAGC GTTTGCAAAA CGACTCGGCA TATTTGGCTG AACACGGTAT 360
TACTGCCGTC TGGATTCCCC CGGCATATAA GGGAACGAGC CAAGCGGATG TGGGCTACGG 420
TGCTTACGAC CTTTATGATT TAGGGGAGTT TCATCAAAAA GGGACGGTTC GGACAAAGTA 480
CGGCACAAAA GGAGAGCTGC AATCTGCGAT CAAAAGTCTT CATTCCCGCG ACATTAACGT 540
TTACGGGGAT GTGGTCATCA ACCACAAAGG CGGCGCTGAT GCGACCGAAG ATGTAACCGC 600
GGTTGAAGTC GATCCCGCTG ACCGCAACCG CGTAATTTCA GGAGAACACC TAATTAAAGC 660
CTGGACACAT TTTCATTTTC CGGGGCGCGG CAGCACATAC AGCGATTTTA AATGGCATTG 720
GTACCATTTT GACGGAACCG ATTGGGACGA GTCCCGAAAG CTGAACCGCA TCTATAAGTT 780
TCAAGGAAAG GCTTGGGATT GGGAAGTTTC CAATGAAAAC GGCAACTATG ATTATTTGAT 840
GTATGCCGAC ATCGATTATG ACCATCCTGA TGTCGCAGCA GAAATTAAGA GATGGGGCAC 900
TTGGTATGCC AATGAACTGC AATTGGACGG TTTCCGTCTT GATGCTGTCA AACACATTAA 960
ATTTTCTTTT TTGCGGGATT GGGTTAATCA TGTCAGGGAA AAAACGGGGA AGGAAATGTT 1020
TACGGTAGCT GAATATTGGC AGAATGACTT GGGCGCGCTG GAAAACTATT TGAACAAAAC 1080
AAATTTTAAT CATTCAGTGT TTGACGTGCC GCTTCATTAT CAGTTCCATG CTGCATCGAC 1140
ACAGGGAGGC GGCTATGATA TGAGGAAATT GCTGAACGGT ACGGTCGTTT CCAAGCATCC 1200
GTTGAAATCG GTTACATTTG TCGATAACCA TGATACACAG CCGGGGCAAT CGCTTGAGTC 1260
GACTGTCCAA ACATGGTTTA AGCCGCTTGC TTACGCTTTT ATTCTCACAA GGGAATCTGG 1320
ATACCCTCAG GTTTTCTACG GGGATATGTA CGGGACGAAA GGAGACTCCC AGCGCGAAAT 1380
TCCTGCCTTG AAACACAAAA TTGAACCGAT CTTAAAAGCG AGAAAACAGT ATGCGTACGG 1440
AGCACAGCAT GATTATTTCG ACCACCATGA CATTGTCGGC TGGACAAGGG AAGGCGACAG 1500
CTCGGTTGCA AATTCAGGTT TGGCGGCATT AATAACAGAC GGACCCGGTG GGGCAAAGCG 1560
AATGTATGTC GGCCGGCAAA ACGCCGGTGA GACATGGCAT GACATTACCG GAAACCGTTC 1620
GGAGCCGGTT GTCATCAATT CGGAAGGCTG GGGAGAGTTT CACGTAAACG GCGGGTCGGT 1680
TTCAATTTAT GTTCAAAGAT AGAAGAGCAG AGAGGACGGA TTTCCTGAAG GAAATCCGTT 1740
TTTTTATTTT GCCCGTCTTA TAAATTTCTT TGATTACATT TTATAATTAA TTTTAACAAA 1800
GTGTCATCAG CCCTCAGGAA GGACTTGCTG ACAGTTTGAA TCGCATAGGT AAGGCGGGGA 1860
TGAAATGGCA ACGTTATCTG ATGTAGCAAA GAAAGCAAAT GTGTCGAAAA TGACGGTATC 1920
GCGGGTGATC AATCATCCTG AGACTGTGAC GGATGAATTG AAAAAGCT 1968 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Ala Asn Leu Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15
Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu 20 25 30
Ala Glu His Gly He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly 35 40 45
Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60
Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80
Gly Glu Leu Gin Ser Ala He Lys Ser Leu His Ser Arg Asp He Asn 85 90 95
Val Tyr Gly Asp Val Val He Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125
He Ser Gly Glu His Leu He Lys Ala Trp Thr His Phe His Phe Pro 130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg He Tyr Lys 165 170 175
Phe Gin Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190
Tyr Asp Tyr Leu Met Tyr Ala Asp He Asp Tyr Asp His Pro Asp Val 195 200 205
Ala Ala Glu He Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gin 210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His He Lys Phe Ser Phe 225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255
Phe Thr Val Ala Glu Tyr Trp Gin Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285
His Tyr Gin Phe His Ala Ala Ser Thr Gin Gly Gly Gly Tyr Asp Met 290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gin Pro Gly Gin Ser Leu Glu 325 330 335
Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe He Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gin Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365
Thr Lys Gly Asp Ser Gin Arg Glu He Pro Ala Leu Lys His Lys He 370 375 380
Glu Pro He Leu Lys Ala Arg Lys Gin Tyr Ala Tyr Gly Ala Gin His 385 390 395 400
Asp Tyr Phe Asp His His Asp He Val Gly Trp Thr Arg Glu Gly Asp 405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu He Thr Asp Gly Pro 420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gin Asn Ala Gly Glu Thr 435 440 445
Trp His Asp He Thr Gly Asn Arg Ser Glu Pro Val Val He Asn Ser 450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser He Tyr 465 470 475 480
Val Gin Arg
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 511 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Met Lys Gin Gin Lys Arg Leu Tyr Ala Arg Leu Leu Thr Leu Leu Phe
1 5 10 15
Ala Leu He Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu 20 25 30
Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro Asn Asp Gly 35 40 45
His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu Ala Glu His Gly 50 55 60
He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly Thr Ser Gin Ala 65 70 75 80
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe His 85 90 95
Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Gly Glu Leu Gin 100 105 110
Ser Ala He Lys Ser Leu His Ser Arg Asp He Asn Val Tyr Gly Asp 115 120 125
Val Val He Asn His Lys Gly Gly Ala Asp Ala Thr Glu Asp Val Thr 130 135 140 Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val He Ser Gly Glu 145 150 155 160
His Leu He Lys Ala Trp Thr His Phe His Phe Pro Gly Arg Gly Ser 165 170 175
Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp 180 185 190
Trp Asp Glu Ser Arg Lys Leu Asn Arg He Tyr Lys Phe Gin Gly Lys 195 200 205
Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn Tyr Asp Tyr Leu 210 215 220
Met Tyr Ala Asp He Asp Tyr Asp His Pro Asp Val Ala Ala Glu He 225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gin Leu Asp Gly Phe 245 250 255
Arg Leu Asp Ala Val Lys His He Lys Phe Ser Phe Leu Arg Asp Trp 260 265 270
Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met Phe Thr Val Ala 275 280 285
Glu Tyr Trp Gin Asn Asp Leu Gly Ala Leu Glu Asn Tyr Leu Asn Lys 290 295 300
Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu His Tyr Gin Phe 305 310 315 320
His Ala Ala Ser Thr Gin Gly Gly Gly Tyr Asp Met Arg Lys Leu Leu 325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser Val Thr Phe Val 340 345 350
Asp Asn His Asp Thr Gin Pro Gly Gin Ser Leu Glu Ser Thr Val Gin 355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe He Leu Thr Arg Glu Ser 370 375 380
Gly Tyr Pro Gin Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Asp 385 390 395 400
Ser Gin Arg Glu He Pro Ala Leu Lys His Lys He Glu Pro He Leu 405 410 415
Lys Ala Arg Lys Gin Tyr Ala Tyr Gly Ala Gin His Asp Tyr Phe Asp 420 425 430
His His Asp He Val Gly Trp Thr Arg Glu Gly Asp Ser Ser Val Ala 435 440 445
Asn Ser Gly Leu Ala Ala Leu He Thr Asp Gly Pro Gly Gly Ala Lys 450 455 460
Arg Met Tyr Val Gly Arg Gin Asn Ala Gly Glu Thr Trp His Asp He 465 470 475 480
Thr Gly Asn Arg Ser Glu Pro Val Val He Asn Ser Glu Gly Trp Gly 485 490 495
Glu Phe His Val Asn Gly Gly Ser Val Ser He Tyr Val Gin Arg 500 505 510
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 520 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Met Arg Gly Arg Gly Asn Met He Gin Lys Arg Lys Arg Thr Val Ser 1 5 10 15
Phe Arg Leu Val Leu Met Cys Thr Leu Leu Phe Val Ser Leu Pro He 20 25 30
Thr Lys Thr Ser Ala Val Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp 35 40 45
Tyr Thr Pro Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp Ala 50 55 60
Glu His Leu Ser Asp He Gly He Thr Ala Val Trp He Pro Pro Ala 65 70 75 80
Tyr Lys Gly Leu Ser Gin Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu 85 90 95
Tyr Asp Leu Gly Glu Phe Gin Gin Lys Gly Thr Val Arg Thr Lys Tyr 100 105 110
Gly Thr Lys Ser Glu Leu Gin Asp Ala He Gly Ser Leu His Ser Arg
115 120 125
Asn Val Gin Val Tyr Gly Asp Val Val Leu Asn His Lys Ala Gly Ala 130 135 140
Asp Ala Thr Glu Asp Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg 145 150 155 160
Asn Gin Glu Thr Ser Glu Glu Tyr Gin He Lys Ala Trp Thr Asp Phe 165 170 175
Arg Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp 180 185 190
Tyr His Phe Asp Gly Ala Asp Trp Asp Glu Ser Arg Lys He Ser Arg 195 200 205
He Phe Lys Phe Arg Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser 210 215 220
Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr 225 230 235 240
Asp His Pro Asp Val Val Ala Glu Thr Lys Lys Trp Gly He Trp Tyr 245 250 255
Ala Asn Glu Leu Ser Leu Asp Gly Phe Arg He Asp Ala Ala Lys His 260 265 270
He Lys Phe Ser Phe Leu Arg Asp Trp Val Gin Ala Val Arg Gin Ala 275 280 285
Thr Gly Lys Glu Met Phe Thr Val Ala Glu Tyr Trp Gin Asn Asn Ala 290 295 300
Gly Lys Leu Glu Asn Tyr Leu Asn Lys Thr Ser Phe Asn Gin Ser Val 305 310 315 320
Phe Asp Val Pro Leu His Phe Asn Leu Gin Ala Ala Ser Ser Gin Gly 325 330 335
Gly Gly Tyr Asp Met Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg 340 345 350
His Pro Glu Lys Ala Val Thr Phe Val Glu Asn His Asp Thr Gin Pro 355 360 365
Gly Gin Ser Leu Glu Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala 370 375 380
Tyr Ala Phe He Leu Thr Arg Glu Ser Gly Tyr Pro Gin Val Phe Tyr 385 390 395 400
Gly Asp Met Tyr Gly Thr Lys Gly Thr Ser Pro Lys Glu He Pro Ser 405 410 415
Leu Lys Asp Asn He Glu Pro He Leu Lys Ala Arg Lys Glu Tyr Ala 420 425 430
Tyr Gly Pro Gin His Asp Tyr He Asp His Pro Asp Val He Gly Trp 435 440 445
Thr Arg Glu Gly Asp Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu 450 455 460
He Thr Asp Gly Pro Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys 465 470 475 480
Asn Ala Gly Glu Thr Trp Tyr Asp He Thr Gly Asn Arg Ser Asp Thr 485 490 495
Val Lys He Gly Ser Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly 500 505 510
Ser Val Ser He Tyr Val Gin Lys 515 520
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 548 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Val Leu Thr Phe His Arg He He Arg Lys Gly Trp Met Phe Leu Leu 1 5 10 15
Ala Phe Leu Leu Thr Ala Ser Leu Phe Cys Pro Thr Gly Arg His Ala 20 25 30
Lys Ala Ala Ala Pro Phe Asn Gly Thr Met Met Gin Tyr Phe Glu Trp 35 40 45
Tyr Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala 50 55 60
Asn Asn Leu Ser Ser Leu Gly He Thr Ala Leu Ser Leu Pro Pro Ala 65 70 75 80
Tyr Lys Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu 85 90 95
Tyr Asp Leu Gly Glu Phe Asn Gin Lys Gly Thr Val Arg Thr Lys Tyr 100 105 110
Gly Thr Lys Ala Gin Tyr Leu Gin Ala He Gin Ala Ala His Ala Ala 115 120 125
Gly Met Gin Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala 130 135 140
Asp Gly Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg 145 150 155 160
Asn Gin Glu He Ser Gly Thr Tyr Gin He Gin Ala Trp Thr Lys Phe 165 170 175
Asp Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
180 185 190
Tyr His Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg 195 200 205
He Tyr Lys Phe Arg Gly He Gly Lys Ala Trp Asp Trp Glu Val Asp 210 215 220
Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met 225 230 235 240
Asp His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr 245 250 255
Val Asn Thr Thr Asn He Asp Gly Phe Arg Leu Asp Gly Leu Lys His 260 265 270
He Lys Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gin 275 280 285
Thr Gly Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp He 290 295 300
Asn Lys Leu His Asn Tyr He Thr Lys Thr Asn Gly Thr Met Ser Leu 305 310 315 320
Phe Asp Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly 325 330 335
Gly Ala Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp 340 345 350
Gin Pro Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Asn Pro 355 360 365
Ala Lys Arg Cys Ser His Gly Arg Pro Trp Phe Lys Pro Leu Ala Tyr 370 375 380
Ala Phe He Leu Thr Arg Gin Glu Gly Tyr Pro Cys Val Phe Tyr Gly 385 390 395 400 Asp Tyr Tyr Gly He Pro Gin Tyr Asn He Pro Ser Leu Lys Ser Lys 405 410 415
He Asp Pro Leu Leu He Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gin 420 425 430
His Asp Tyr Leu Asp His Ser Asp He He Gly Trp Thr Arg Glu Gly 435 440 445
Val Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu He 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 He 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 He Ala Arg Pro He 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
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Ala Asn Leu Asn Gly Thr Leu Met Gin Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15
Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp Ser Ala Tyr Leu 20 25 30
Ala Glu His Gly He Thr Ala Val Trp He Pro Pro Ala Tyr Lys Gly 35 40 45
Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60
Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80
Gly Glu Leu Gin Ser Ala He Lys Ser Leu His Ser Arg Asp He Asn 85 90 95
Val Tyr Gly Asp Val Val He Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110
Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 He Ser Gly Glu His Leu He Lys Ala Trp Thr His Phe His Phe Pro 130 135 140
Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg He Tyr Lys 165 170 175
Phe Gin Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190
Tyr Asp Tyr Leu Thr Tyr Ala Asp He Asp Tyr Asp His Pro Asp Val 195 200 205
Ala Ala Glu He Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gin 210 215 220
Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His He Lys Phe Ser Phe 225 230 235 240
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255
Phe Thr Val Ala Glu Tyr Trp Gin Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270
Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285
His Tyr Gin Phe His Ala Ala Ser Thr Gin Gly Gly Gly Tyr Asp Met 290 295 300
Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gin Pro Gly Gin Ser Leu Glu 325 330 335
Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe He Leu 340 345 350
Thr Arg Glu Ser Gly Tyr Pro Gin Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365
Thr Lys Gly Asp Ser Gin Arg Glu He Pro Ala Leu Lys His Lys He 370 375 380
Glu Pro He Leu Lys Ala Arg Lys Gin Tyr Ala Tyr Gly Ala Gin His 385 390 395 400
Asp Tyr Phe Asp His His Asp He Val Gly Trp Thr Arg Glu Gly Asp 405 410 415
Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu He Thr Asp Gly Pro 420 425 430
Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gin Asn Ala Gly Glu Thr 435 440 445
Trp His Asp He Thr Gly Asn Arg Ser Glu Pro Val Val He Asn Ser 450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser He Tyr 465 470 475 480
Val Gin Arg (2) INFORMATION FOR SEQ ID NO:37:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 aπαno acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) 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 15
Trp Tyr Met Pro Asn Asp Gly Gin His Trp Lys Arg Leu Gin Asn Asp 20 25 30
Ser Ala Tyr Leu Ala Glu His Gly He Thr Ala Val Trp He Pro Pro 35 40 45
Ala Tyr Lys Gly Thr Ser Gin Ala Asp Val Gly Tyr Gly Ala Tyr Asp 50 55 60
Leu Tyr Asp Leu Gly Glu Phe His Gin Lys Gly Thr Val Arg Thr Lys 65 70 75 80
Tyr Gly Thr Lys Gly Glu Leu Gin Ser Ala He Lys Ser Leu His Ser 85 90 95
Arg Asp He Asn Val Tyr Gly Asp Val Val He 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 He Ser Gly Glu His Leu He 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 He 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 He Asp Tyr Asp 195 200 205
His Pro Asp Val Ala Ala Glu He 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 He 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 Asp Val Pro Leu His Tyr Gin Phe His Ala Ala Ser Thr Gin Gly Gly 290 295 300
Gly Tyr Asp Met Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His 305 310 315 320
Pro Leu Lys Ser Val Thr Phe Val Asp Asn His Asp Thr Gin Pro Gly 325 330 335
Gin Ser Leu Glu Ser Thr Val Gin Thr Trp Phe Lys Pro Leu Ala Tyr 340 345 350
Ala Phe He Leu Thr Arg Glu Ser Gly Tyr Pro Gin Val Phe Tyr Gly 355 360 365
Asp Met Tyr Gly Thr Lys Gly Asp Ser Gin Arg Glu He Pro Ala Leu 370 375 380
Lys His Lys He Glu Pro He Leu Lys Ala Arg Lys Gin Tyr Ala Tyr 385 390 395 400
Gly Ala Gin His Asp Tyr Phe Asp His His Asp He Val Gly Trp Thr 405 410 415
Arg Glu Gly Asp Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu He 420 425 430
Thr Asp Gly Pro Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gin Asn 435 440 445
Ala Gly Glu Thr Trp His Asp He Thr Gly Asn Arg Ser Glu Pro Val 450 455 460
Val He Asn Ser Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser 465 470 475 480
Val Ser He Tyr Val Gin Arg 485
(2) INFORMATION FOR SEQ ID NO:38:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Met Lys Gin Gin Lys Arg Leu Thr Ala Arg Leu Leu Thr Leu Leu Phe 1 5 10 15
Ala Leu He Phe Leu Leu Pro His Ser Ala Ala Ala Ala Ala Asn Leu 20 25 30
(2) INFORMATION FOR SEQ ID NO:39:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ( 11 ) MOLECULE TYPE : protein
(xi ) SEQUENCE DESCRI PTION: SEQ ID NO : 39 :
Met Arg Ser Lys Thr Leu Trp He Ser Leu Leu Phe Ala Leu Thr Leu
1 5 10 15
He Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Gly Lys 20 25 30
Ser
(2) INFORMATION FOR SEQ ID NO:40:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Met Arg Ser Lys Thr Leu Trp He Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15
He Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Ala Ala 20 25 30
Ala Ala Asn 35
(2) INFORMATION FOR SEQ ID NO:41:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Met Arg Ser Lys Thr Leu Trp He Ser Leu Leu Phe Ala Leu Thr Leu 1 5 10 15
He Phe Thr Met Ala Phe Ser Asn Met Ser Ala Gin Ala Ala Asn Leu 20 25 30
(2) INFORMATION FOR SEQ ID NO:42:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CACCTAATTA AAGCTTTCAC ACATTTTCAT TTT 33
(2) INFORMATION FOR SEQ ID NO: 3:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: CACCTAATTA AAGCTTACAC ACATTTTCAT TTT 33
(2) INFORMATION FOR SEQ ID NO:44:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CCGCGTAATT TCCGGAGAAC ACCTAATTAA AGCCGCAACA CATTTTCATT TTCCCGGGCG 60 CGGCAG 66
(2) INFORMATION FOR SEQ ID NO:45:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: CCGGAGAACA CCTAATTAAA GCCCTAACAC ATTTTCATTT TC 42
(2) INFORMATION FOR SEQ ID NO: 46:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CCGGAGAACA CCTAATTAAA GCCCACACAC ATTTTCATTT TC 42
(2) INFORMATION FOR SEQ ID NO:47:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: CCGGAGAACA CCTAATTAAA GCCTGCACAC ATTTTCATTT TC 42
(2) INFORMATION FOR SEQ ID NO: 48:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 48: GATGCAGTAT TTCGAACTGG TATA 24
(2) INFORMATION FOR SEQ ID NO: 49:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: TGCCCAATGA TGGCCAACAT TGGAAG 26
(2) INFORMATION FOR SEQ ID NO:50:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 50: CGAATGGTAT GCTCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO:51:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CGAATGGTAT CGCCCCAATG ACGG 2
(2) INFORMATION FOR SEQ ID NO:52:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CGAATGGTAT AATCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:53:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: CGAATGGTAT GATCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:54:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CGAATGGTAT CACCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:55: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: CGAATGGTAT AAACCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGAATGGTAT CCGCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:57:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CGAATGGTAT TCTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CGAATGGTAC ACTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:59:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CGAATGGTAT GTTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO: 60:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: CGAATGGTAT TGTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:61:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: CGAATGGTAT CAACCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO: 62:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:62: CGAATGGTAT GAACCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO: 63:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( ll ) MOLECULE TYPE : DNA ( genomic ) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CGAATGGTAT GGTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:64:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:64: CGAATGGTAT ATTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:65:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: CGAATGGTAT TTTCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:66:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CGAATGGTAC TGGCCCAATG ACGG 24
(2) INFORMATION FOR SEQ ID NO:67:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: CGAATGGTAT TATCCCAATG ACGG 24 (2) INFORMATION FOR SEQ ID NO: 68:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 68: CCGTCATTGG GACTACGTAC CATT 24

Claims

WHAT IS CLAIMED IS:
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 by the substitution of a methionine at a position equivalent to M+197 in B. li cheniformi s 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.
2. An improved laundry detergent composition of Claim 1 wherein the cleaning composition is a liquid composition.
3. An improved laundry detergent composition of Claim 1 wherein the mutant alpha-amylase is selected from the group consisting of M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T and M15T/W138Y/M197T.
4. An improved laundry detergent composition of Claim 1 further comprising a mutant protease that is the expression product of a mutated DNA sequence encoding a protease, the mutated DNA sequence being derived from a precursor protease by the substitution of a methionine at a position equivalent to M+222 in Bacill us amyloliquefaciens protease.
5. An improved laundry detergent composition of Claim 4 wherein the mutant protease comprises a substitution selected from the group of amino acids consisting of alanine, cysteine and serine.
6. An improved laundry detergent composition of Claim 4 comprising an alpha-amylase mutant selected from the group consisting of M15T/M197T, M15S/M197T, W138Y/M197T, M15S/W138Y/M197T and M15T/W138Y/M197T, and a protease mutant selected from the group consisting of M222C, M222S and M222A.
7. An improved laundry detergent composition of Claim 2, wherein the liquid detergent is at a pH of between about 6.0 and 10.0.
8. An improved laundry detergent composition according to claim 1, wherein said laundry detergent contains bleach.
9. An improved laundry detergent composition according to claim
8, wherein said laundry detergent has a pH of above about 10.
10. An improved laundry detergent composition according to claim
9, wherein said laundry detergent is granular.
EP96909854A 1995-03-24 1996-03-22 An improved laundry detergent composition comprising amylase Withdrawn EP0815193A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US409771 1989-09-20
US40977195A 1995-03-24 1995-03-24
PCT/US1996/004029 WO1996030481A1 (en) 1995-03-24 1996-03-22 An improved laundry detergent composition comprising amylase

Publications (1)

Publication Number Publication Date
EP0815193A1 true EP0815193A1 (en) 1998-01-07

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Country Status (10)

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EP (1) EP0815193A1 (en)
JP (1) JPH11502562A (en)
KR (1) KR19980702783A (en)
AU (1) AU718509B2 (en)
BR (1) BR9607751A (en)
CA (1) CA2216316A1 (en)
MX (1) MX9706823A (en)
NO (1) NO974402L (en)
NZ (1) NZ305257A (en)
WO (1) WO1996030481A1 (en)

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NO974402D0 (en) 1997-09-23
AU5322696A (en) 1996-10-16
JPH11502562A (en) 1999-03-02
MX9706823A (en) 1997-11-29
AU718509B2 (en) 2000-04-13
NO974402L (en) 1997-11-19
NZ305257A (en) 1999-01-28
CA2216316A1 (en) 1996-10-03
WO1996030481A1 (en) 1996-10-03
BR9607751A (en) 1998-06-23
KR19980702783A (en) 1998-08-05

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