WO1996005295A2 - An improved cleaning composition - Google Patents

An improved cleaning composition Download PDF

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Publication number
WO1996005295A2
WO1996005295A2 PCT/US1995/010426 US9510426W WO9605295A2 WO 1996005295 A2 WO1996005295 A2 WO 1996005295A2 US 9510426 W US9510426 W US 9510426W WO 9605295 A2 WO9605295 A2 WO 9605295A2
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Prior art keywords
amylase
alpha
amylases
m197t
mutant
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PCT/US1995/010426
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French (fr)
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WO1996005295A3 (en
Inventor
Christopher C. Barnett
Colin Mitchinson
Scott D. Power
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Genencor International, Inc.
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Priority to US28935194A priority Critical
Priority to US08/289,351 priority
Application filed by Genencor International, Inc. filed Critical Genencor International, Inc.
Publication of WO1996005295A2 publication Critical patent/WO1996005295A2/en
Publication of WO1996005295A3 publication Critical patent/WO1996005295A3/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405Glucanases
    • C12N9/2408Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411Amylases
    • C12N9/2414Alpha-amylase (3.2.1.1.)
    • C12N9/2417Alpha-amylase (3.2.1.1.) from microbiological source
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11DDETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16Organic compounds
    • C11D3/38Products with no well-defined composition, e.g. natural products
    • C11D3/386Preparations containing enzymes, e.g. protease, amylase
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11DDETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/39Organic or inorganic per-compounds
    • C11D3/3942Inorganic per-compounds
    • CCHEMISTRY; METALLURGY
    • C11ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11DDETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/39Organic or inorganic per-compounds
    • C11D3/3947Liquid compositions

Abstract

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

Description

AN IMPROVED CLEANING COMPOSITION

Field of the Invention

The present invention relates to novel alpha-amylase mutants having an amino acid sequence not found in nature, such mutants having an amino acid sequence wherein one or more amino acid residue (s) of a precursor alpha-amylase, specifically any oxidizable amino acid, have been substituted with a different amino acid. The mutant enzymes of the present invention exhibit altered stability/activity profiles including but not limited to altered oxidative stability, altered pH performance profile, altered specific activity and/or altered thermostability.

Background of the Invention

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolase, EC3.2.1.1) hydrolyze internal alpha-1,4-glucosidic linkages in starch largely at random, to produce smaller molecular weight malto-dextrins.

Alpha-amylases are of considerable commercial value, being used in the initial stages (liquefaction) of starch processing; in alcohol production; as cleaning agents in detergent matrices; and in the textile industry for starch desizing. Alpha-amylases are produced by a wide variety of microorganisms including Bacillus and

Aspergillus , with most commercial amylases being produced from bacterial sources such as B. lichenifozmis, B. amyloliquefaciens, B. subtilis, or B. stearothermophilus. In recent years the preferred enzymes in commercial use have been those from B. licheniformis because of their heat stability and performance, at least at neutral and mildly alkaline pH's.

Previously there have been studies using recombinant DNA techniques to explore which residues are important for the catalytic activity of amylases and/or to explore the effect of modifying certain amino acids within the active site of various amylases (Vihinen, M. et al . (1990) J. Bichem. 107:267-272; Holm, L. et al. (1990) Protein

Engineering 3:181-191; Takase, K. et al. (1992) Biochemica et

Biophysica Acta, 1120:281-288; Matsui, I. et al. (1992) Febs Letters Vol. 310, No. 3, pp. 216-218); which residues are important for thermal stability (Suzuki, Y. et al. (1989) J. Biol. Chem.

264:18933-18938); and one group has used such methods to introduce mutations at various histidine residues in a B. licheniformis amylase, the rationale for making substitutions at histidine residues was that B. licheniformis amylase (known to be

thermostable) when compared to other similar -Bacillus amylases, has an excess of histidines and, therefore, it was suggested that replacing a histidine could affect the thermostability of the enzyme (Declerck, N. et al. (1990) J. Biol. Chem. 265:15481-15488; FR 2 665 178-A1; Joyet, P. et al. (1992) Bio/Technology 10:1579-1583).

It has been found that alpha-amylase is inactivated by hydrogen peroxide and other oxidants at pH's between 4 and 10.5 as described in the examples herein. Commercially, alpha-amylase enzymes can be used under dramatically different conditions such as both high and low pH conditions, depending on the commercial application. For example, alpha-amylases may be used in the liquefaction of starch, a process preferably performed at a low pH (pH <5.5). On the other hand, amylases may be used in commercial dish care or laundry detergents, which often contain oxidants such as bleach or peracids, and which are used in much more alkaline conditions.

In order to alter the stability or activity profile of amylase enzymes under varying conditions, it has been found that selective replacement, substitution or deletion of oxidizable amino acids, such as a methionine, tryptophan, tyrosine, histidine or cysteine, results in an altered profile of the variant enzyme as compared to its precursor. Because currently commercially available amylases are not acceptable (stable) under various conditions, there is a need for an amylase having an altered stability and/or activity profile. This altered stability (oxidative, thermal or pH

performance profile) can be achieved while maintaining adequate enzymatic activity, as compared to the wild-type or precursor enzyme. The characteristic affected by introducing such mutations may be a change in oxidative stability while maintaining thermal stability or vice versa . Additionally, the substitution of

different amino acids for an oxidizable amino acids in the alpha- amylase precursor sequence or the deletion of one or more oxidizable amino acid(s) may result in altered enzymatic activity at a pH other than that which is considered optimal for the precursor alpha- amylase. In other words, the mutant enzymes of the present invention may also have altered pH performance profiles, which may be due to the enhanced oxidative stability of the enzyme. Summary of the invention

The present invention relates to novel alpha-amylase mutants that are the expression product of a mutated DNA sequence encoding an alpha-amylase, the mutated DNA sequence being derived from a precursor alpha-amylase by the deletion or substitution

(replacement) of one or more oxidizable amino acid. In one

preferred embodiment of the present invention the mutant result from substituting a different amino acid for one or more methionine residue (s) in the precursor alpha-amylase. In another embodiment of the present invention the mutants comprise a substitution of one or more tryptophan residue alone or in combination with the

substitution of one or more methionine residue in the precursor alpha-amylase. Such mutant alpha-amylases, in general, are obtained by in vi tro modification of a precursor DNA sequence encoding a naturally occurring or recombinant alpha-amylase to encode the substitution or deletion of one or more amino acid residues in a precursor amino acid sequence.

Preferably the substitution or deletion of one or more amino acid in the amino acid sequence is due to the replacement or deletion of one or more methionine, tryptophan, cysteine, histidine or tyrosine residues in such sequence, most preferably the residue which is changed is a methionine residue. The oxidizable amino acid residues may be replaced by any of the other 20 naturally occurring amino acids. If the desired effect is to alter the oxidative stability of the precursor, the amino acid residue may be substituted with a non- oxidizable amino acid (such as alanine, arginine, asparagine, aspartic acid, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, or valine) or another oxidizable amino acid (such as cysteine,

methionine, tryptophan, tyrosine or histidine, listed in order of most easily oxidizable to less readily oxidizable). Likewise, if the desired effect is to alter thermostability, any of the other 20 naturally occurring amino acids may be substituted (i.e., cysteine may be substituted for methionine). Preferred mutants comprise the substitution of a methionine residue equivalent to any of the methionine residues found in B.

licheniformis alpha-amylase (+8, +15, +197, +256, +304, +366 and +438). Most preferably the methionine to be replaced is a

methionine at a position equivalent to position +197 or +15 in B. licheniformis alpha-amylase. Preferred substitute amino acids to replace the methionine at position +197 are alanine (A), isoleucine (I), threonine (T) or cysteine (C). The preferred substitute amino acids at position +15 are leucine (L), threonine (T), asparagine (N), aspartate (D), serine (S), valine (V) and isoleucine (I), although other substitute amino acids not specified above may be useful. Two specifically preferred mutants of the present invention are M197T and M15L.

Another embodiment of this invention relates to mutants comprising the substitution of a tryptophan residue equivalent to any of the tryptophan residues found in B. licheniformis alpha-amylase (see Fig. 2). Preferably the tryptophan to be replaced is at a position equivalent to +138 in B. licheniformis alpha-amylase. A mutation (substitution) at a tryptophan residue may be made alone or in combination with mutations at other oxidizable amino acid residues. Specifically, it may be advantageous to modify by substitution at least one tryptophan in combination with at least one methionine (for example, the double mutant +138/+197).

The alpha-amylase mutants of the present invention, in general, exhibit altered oxidative stability in the presence of hydrogen peroxide and other oxidants such as bleach or peracids, or, more specific, milder oxidants such as chloramine-T . Mutant enzymes having enhanced oxidative stability will be useful in extending the shelf life and bleach, perborate, percarbonate or peracid

compatibility of amylases used in cleaning products. Similarly, reduced oxidative stability may be 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 end use of the mutant alpha-amylase.

In addition to starch processing and cleaning applications, variant amylases of the present invention may be used in any application in which known amylases are used, for example, variant amylases can be used in textile processing, food processing, etc. Specifically, it is contemplated that a variant enzyme such as M197C, which is easily inactivated by oxidation, would be useful in a process where it is desirable to completely remove amylase activity at the end of the process, for example, in frozen food processing applications.

The preferred alpha-amylase mutants of the present invention are derived from a Bacillus strain such as B. licheniformis, B.

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

In another aspect of the present invention there is provided a novel form of the alpha-amylase normally produced by B. licheniformis . This novel form, designated as the A4 form, has an additional four alanine residues at the N-terminus of the secreted amylase. (Fig. 4b.) Derivatives or mutants of the A4 form of alpha-amylase are encompassed within the present invention. By derivatives or mutants of the A4 form, it is meant that the present invention comprises the A4 form alpha-amylase containing one or more additional mutations such as, for example, mutation (substitution, replacement or deletion) of one or more oxidizable amino acid(s).

In a composition embodiment of the present invention there are provided detergent compositions, liquid, gel or granular, comprising the alpha-amylase mutants described herein. Particularly preferred are detergent compositions comprising a +197 position mutant either alone or in combination with other enzymes such as endoglycosidases, cellulases, proteases, lipases or other amylase enzymes.

Additionally, it is contemplated that the compositions of the present invention may include an alpha-amylase mutant having more than one site-specific mutation.

In yet another composition embodiment of the present invention there are provided compositions useful in starch processing and particularly starch liquefaction. The starch liquefaction

compositions of the present invention preferably comprise an alpha- amylase mutant having a substitution or deletion at position M15. Additionally, it is contemplated that such compositions may comprise additional components as known to those skilled in the art,

including, for example, antioxidants, calcium, ions, etc.

In a process aspect of the present invention there are provided methods for liquefying starch, and particularly granular starch slurries, from either a wet or dry milled process. Generally, in the first step of the starch degradation process, the starch slurry is gelatinized by heating at a relatively high temperature (up to about 110°C). After the starch slurry is gelatinized it is

liquefied and dextrinized using an alpha-amylase. The conditions for such liquefaction are described in commonly assigned US patent applications 07/785,624 and 07/785,623 and US Patent 5,180,669, the disclosure of which are incorporated herein by reference. The present method for liquefying starch comprises adding to a starch slurry an effective amount of an alpha-amylase of the present invention, alone or in combination with additional excipients such as an antioxidant, and reacting the slurry for an appropriate time and temperature to liquefy the starch.

A further aspect of the present invention comprises the DNA encoding the mutant alpha-amylases of the present invention (including A4 form and mutants thereof) and expression vectors encoding the DNA as well as host cells transformed with such expression vectors.

Brief Description of the Drawing

Fig. 1 shows the DNA sequence of the gene for alpha-amylase from B. licheniformis (NCIB8061), Seq ID No 31, and deduced translation product as described in Gray, G. et al. (1986) J. Bacter. 166:635- 643.

Fig. 2 shows the amino acid sequence of the mature alpha-amylase enzyme from B. licheniformis (NCIB8061), Seq ID No 32.

Fig. 3 shows an alignment of primary structures of Bacillus alpha- amylases. The B . licheniformis amylase (Am-Lich), Seq ID No 33, is described by Gray, G. et al. (1986) J. Bact. 166:635-643; the B. amyl oliquefaciens amylase (Am-Amylo), Seq ID No 34, is described by Takkinen, K. et al. (1983) J. Biol. Chem. 258:1007-1013; and the B. stearothermophilus (Am-Stearo), Seq ID No 35, is described by Ihara, H. et al. (1985) J. Biochem. 98:95-103.

Fig. 4a shows the amino acid sequence of the mature alpha-amylase variant M197T, Seq ID No 36.

Fig. 4b shows the amino acid sequence of the A4 form of alpha- amylase from B. licheniformis NCIB8061, Seq ID No 37. Numbering is from the N-terminus, starting with the four additional alanines.

Fig. 5 shows plasmid pA4BL wherein BLAA refers to B. licheniformis alpha-amylase gene, Pstl 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 H2O2 at pH 5.0, 25°C.

Fig. 7b shows inactivation of certain alpha-amylases (Spezyme® AA20, M197T) with 0.88M H2O2 at pH 10.0, 25°C.

Fig. 7c shows inactivation of certain alpha-amylases (Spezyme® AA20, M15L) with 0.88M H2O2 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. licheniformis wild-type.

Fig. 17 shows the inactivation of B. licheniformis 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. licheniformis 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. licheniformis alpha-amylase (AA20 at 0.75 mg/ml) with chloramine-T at pH 5.0 as compared to double variants M197T/W138F (0.64 mg/ml) and M197T/W138Y (0.60 mg/ml).

Fig. 20 shows the stability testing results of various alpha-amylase multiple mutants incorporated in automatic dish detergent (ADD) formulations at temperatures from room temperature increased to 65°C.

Fig. 21 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at room temperature over 0-30 days, as determined by percent activity remaining over time. Fig. 22 shows the stability of certain amylase mutants (compared to wild-type) in an automatic dish detergent at 38°C (100°F) with 80% relative humidity over 0-30 days.

Detailed Description of the 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. The mutant enzymes of the present invention may also have an altered pH profile and/or altered thermal stability which may be due to the enhanced oxidative stability of the enzyme at low or high pH's.

Alpha-amylase as used herein includes naturally occurring amylases as well as recombinant amylases. Preferred amylases in the present invention are alpha-amylases derived from B. licheniformis or B. stearothermophilus, including the A4 form of alpha-amylase derived from B. licheniformis as described herein, as well as fungal alpha- amylases such as those derived from Aspergillus (i.e., A. oryzae and A . niger) .

Recombinant alpha-amylases refers to an alpha-amylase in which the DNA sequence encoding the naturally occurring alpha-amylase is modified to produce a mutant DNA sequence which encodes the

substitution, insertion or deletion of one or more amino acids in the alpha-amylase sequence. Suitable modification methods are disclosed herein, and also in commonly owned US Patents 4,760,025 and 5,185,258, the disclosure of which are incorporated herein by reference.

Homologies have been found between almost all endo-amylases

sequenced to date, ranging from plants, mammals, and bacteria

(Nakajima, R.T. et al. (1986) Appl . Microbiol. Biotechnol. 23:355- 360; Rogers, J.C. (1985) Biochem. Biophys. Res. Commun. 128:470- 476). There are four areas of particularly high homology in certain Bacillus amylases, as shown in Fig. 3, wherein the underlined sections designate the areas of high homology. Further, sequence alignments have been used to map the relationship between Bacillus endo-amylases (Feng, D.F. and Doolittle, R.F. (1987) J. Molec. Evol. 35:351-360). The relative sequence homology between B.

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

amyloliquefaciens amylases is about 81%, as per Holm, L. et al., supra . While sequence homology is important, it is generally recognized that structural homology is also important in comparing amylases or other enzymes. For example, structural homology between fungal amylases and bacterial (Bacillus) amylase have been suggested and, therefore, fungal amylases are encompassed within the present invention.

An alpha-amylase mutant has an amino acid sequence which is derived from the amino acid sequence of a precursor alpha-amylase. The precursor alpha-amylases include naturally occurring alpha-amylases and recombinant alpha-amylases (as defined). The amino acid sequence of the alpha-amylase mutant is derived from the precursor alpha-amylase amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the precursor DNA sequence which encodes the amino acid sequence of the precursor alpha-amylase rather than manipulation of the precursor alpha-amylase enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein and in commonly owned US patent 4,760,025 and 5,185,258.

Specific residues corresponding to positions M197, M15 and W138 of Bacillus licheniformis alpha-amylase are identified herein for substitution or deletion, as are all methionine, histidine,

tryptophan, cysteine and tyrosine positions. The amino acid position number (i.e., +197) refers to the number assigned to the mature Bacillus licheniformis alpha-amylase sequence presented in Fig. 2. The invention, however, is not limited to the mutation of this particular mature alpha-amylase (B. licheniformis) but extends to precursor alpha-amylases containing amino acid residues at positions which are equivalent to the particular identified residue in B. licheniformis alpha-amylase. A residue (amino acid) of a precursor alpha-amylase is equivalent to a residue of B. licheniformis alpha-amylase if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. licheniformis alpha-amylase (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. licheniformis alpha-amylase primary sequence and particularly to a set of residues known to be invariant to all alpha-amylases for which sequence is known, as seen in Fig. 3. It is possible also to determine equivalent residues by tertiary structure: crystal structures have been reported for porcine pancreatic alpha-amylase (Buisson, G. et al. (1987) EMBO J.6 : 3909-3916); Taka-amylase A from Aspergillus oryzae (Matsuura, Y. et al. (1984) J. Biochem. (Tokyo) 95:697-702); and an acid alpha-amylase from A . niger (Boel, E. et al. (1990) Biochemistry 29:6244-6249), with the former two

structures being similar. There are no published structures for Bacillus alpha-amylases, although there are predicted to be common super-secondary structures between glucanases (MacGregor, E.A. & Svensson, B. (1989) Biochem. J. 259:145-152) and a structure for the B. stearothermophilus enzyme has been modeled on that of 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 licheniformis numbering, His105; Arg229;

Asp231; His235; Glu261 and Asp328.

Expression vector as used herein refers to a DNA construct

containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome-binding sites, and sequences which control termination of transcription and translation. A preferred promoter is the B. subtilis aprEE promoter. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, plasmid and vector are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

Host strains (or cells) useful in the present invention generally are procaryotic or eucaryotic hosts and include any transformable microorganism in which the expression of alpha-amylase can be achieved. Specifically, host strains of the same species or genus from which the alpha-amylase is derived are suitable, such as a Bacillus strain. Preferably an alpha-amylase negative Bacillus strain (genes deleted) and/or an alpha-amylase and protease deleted Bacillus strain such as Bacillus subtilis strain BG2473

(ΔamyE, Δapr, Δnpr) is used. Host cells are transformed or

transfected with vectors constructed using recombinant DNA

techniques. Such transformed host cells are capable of either replicating vectors encoding the alpha-amylase and its variants (mutants) or expressing the desired alpha-amylase.

Preferably the mutants of the present invention are secreted into the culture medium during fermentation. Any suitable signal sequence, such as the aprE signal peptide, can be used to achieve secretion.

Many of the alpha-amylase mutants of the present invention are useful in formulating various detergent compositions, particularly certain dish care cleaning compositions, especially those cleaning compositions containing known oxidants. Alpha-amylase mutants of the invention can be formulated into known powdered, liquid or gel detergents having pH between 6.5 to 12.0. Suitable granular composition may be made as described in commonly owned US patent applications 07/429,881, 07/533,721 and 07/957,973, all of which are incorporated herein by reference. These detergent cleaning

compositions can also contain other enzymes, such as known

proteases, lipases, cellulases, endoglycosidases or other amylases, as well as builders, stabilizers or other excipients known to those skilled in the art. These enzymes can be present as co-granules or as blended mixes or in any other manner known to those skilled in the art. Furthermore, it is contemplated by the present invention that multiple mutants may be useful in cleaning or other

applications. For example, a mutant enzyme having changes at both +15 and +197 may exhibit enhanced performance useful in a cleaning product or a multiple mutant comprising changes at +197 and +138 may have improved performance. Specifically preferred mutant enzymes for use in cleaning products, and particularly dish care

formulations, include but are not limited to M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T; and M15T/W138Y/M197T.

Another embodiment of the present invention comprises the

combination of the mutant alpha-amylase enzymes described herein in combination with other enzymes (i.e., proteases, lipases,

cellulases, etc.), and preferably oxidatively stable proteases.

Suitable oxidatively stable proteases include genetically engineered proteases such as those described in US Re 34606, incorporated herein by reference, as well as commercially available enzymes such as DURAZYM (Novo Nordisk), MAXAPEM (Gist-brocades) and PURAFECT OXP (Genencor International, Inc.). Suitable methods for making such protease mutants (oxidatively stable proteases), and particularly such mutants having a substitution for the methionine at a position equivalent to M222 in B. amyloliquefaci ens, are described in US Re 34606. Suitable methods for determining "equivalent" positions in other subtilisins are provided in Re 34606, EP 257,446 and USSN 212,291, which are incorporated herein by reference.

As described previously, alpha-amylase mutants of the present invention may also be useful in the liquefaction of starch. Starch liquefaction, particularly granular starch slurry liquefaction, is typically carried out at near neutral pH's and high temperatures. As described in commonly owned US applications 07/788,624 and

07/785,623 and US Patent 5,180,669, it appears that an oxidizing agent or inactivating agent of some sort is also present in typical liquefaction processes, which may affect the enzyme activity; thus, in these related patent applications an antioxidant is added to the process to protect the enzyme.

Based on the conditions of a preferred liquefaction process, as described in commonly owned US applications 07/788,624 and

07/785,623 and US Patent 5,180,669, namely low pH, high temperature and potential oxidation conditions, preferred mutants of the present invention for use in liquefaction processes comprise mutants exhibiting altered pH performance profiles (i.e., low pH profile, pH <6 and preferably pH <5.5), and/or altered thermal stability (i.e., high temperature, about 90°-110°C), and/or altered oxidative stability (i.e., enhanced oxidative stability).

Thus, an improved method for liquefying starch is taught by the present invention, the method comprising liquefying a granular starch slurry from either a wet or dry milling process at a pH from about 4 to 6 by adding an effective amount of an alpha-amylase mutant of the present invention to the starch slurry; optionally adding an effective amount of an antioxidant or other excipient to the slurry; and reacting the slurry for an appropriate time and temperature to liquefy the starch.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims. Abbreviations used herein, particularly three letter or one letter notations for amino acids are described in Dale, J.W., Molecular Genetics of Bacteria, John Wiley & Sons, (1989) Appendix B.

Experimental

Example 1

Substitutions for the Methionine Residues in

B . l i cheni formis Alpha-Amy lase

The alpha-amylase gene (Fig. 1) was cloned from B. licheniformis NCIB8061 obtained from the National Collection of Industrial

Bacteria, Aberdeen, Scotland (Gray, G. et al. (1986) J. Bacteriology 166:635-643). The 1.72kb 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:

Figure imgf000016_0001
designed to contain the B. amyloliquefaciens subtilisin

transcriptional terminator (Wells et al. (1983) Nucleic Acid

Research 11:7911-7925).

Site-directed mutagenesis by oligonucleotides used essentially the protocol of Zoller, M. et al. (1983) Meth. Enzymol. 100:468-500: briefly, 5'-phosphorylated oligonucleotide primers were used to introduce the desired mutations on the M13 single-stranded DNA template using the oligonucleotides listed in Table I to substitute for each of the seven methionines found in B. licheniformis alpha- amylase. Each mutagenic oligonucleotide also introduced a

restriction endonuclease site to use as a screen for the linked mutation.

Figure imgf000018_0001

Bold letter indicate base changes introduced by oligonucleotide.

Codon changes indicated in the form M8A, where methionine (M) at position +8 has been changed to alanine (A).

Underlining indicates restriction endonuclease site introduced by oligonucleotide.

The heteroduplex was used to transfect E. coli mutL cells (Kramer et al. (1984) Cell 38:879) and, after plaque-purification, clones were analyzed by restriction analysis of the RFl's. Positives were confirmed by dideoxy sequencing (Sanger et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) and the Pstl-Sstl fragments for each 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 pJHIO1 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. licheniformis alpha-amylase gave pA4BL (Fig. 5) having the resulting aprE signal peptide-amylase junction as shown in Fig. 6.

Transformation Into B . subti l i s

pA4BL is a plasmid able to replicate in E. coli and integrate into the B. subtilis chromosome. Plasmids containing different variants were transformed into B. subtilis (Anagnostopoulos, C. and Spizizen, J. (1961) J. Bacter. 81:741-746) and integrated into the chromosome at the aprE locus by a Campbell-type mechanism (Young, M. (1984) J. Gen. Microbiol. 130:1613-1621). The Bacillus subtilis strain BG2473 was a derivative of 1168 which had been deleted for amylase (ΔamyE) and two proteases (Lapr, Lnpr) (Stahl, M.L. and Ferrari, E., J.

Bacter. 158:411-418 and US Patent 5,264,366, incorporated herein by reference). After transformation the sacϋ32 (Hy) (Henner, D.J. et al. (1988) J. Bacter. 170:296-300) mutation was introduced by PBS-1 mediated transduction (Hoch, J.A. (1983) 154:1513-1515).

N-terminal analysis of the amylase expressed from pA4BL in B.

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 eaqperiments the correctly processed forms of the licheniformis 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:

Figure imgf000020_0001

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. licheniformis alpha-amylase.

Example 2

Oxidative Sensitivity of Methionine Variants B. licheniformis alpha-amylase, such as Spezyme® AA20 (commercially available from Genencor International, Inc.), is inactivated rapidly in the presence of hydrogen peroxide (Fig. 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. licheniformis 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:

Figure imgf000021_0001
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.

r o

Figure imgf000022_0001

Figure imgf000023_0001

The cassettes were designed to destroy the EcoRV site upon ligation, thus plasmids from E. coli transformants were screened for loss of this unique site. In addition, the common bottom strand of the cassette contained a frame-shift and encoded a NsiI site, thus transformants derived from this strand could be eliminated by screening for the presence of the unique Nsil site and would not be expected, in any case, to lead to expression of active amylase.

Positives by restriction analysis were confirmed by sequencing and transformed in B. subtilis for expression in shake-flask cultures (Fig. 9). The specific activity of certain of the M197X mutants was then determined using a soluble substrate assay. The data generated using the following assay methods are presented below in Table III.

Soluble Substrate Assay: A rate assay was developed based on an. end-point assay kit supplied by Megazyme (Aust.) Pty. Ltd.: Each vial of substrate (p-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10ml of sterile water, followed by a 1 to 4 dilution in assay buffer (50mM maleate buffer, pH 6.7, 5mM calcium chloride, 0.002% Tween20). Assays were performed by adding lOut of amylase to 790u< of the substrate in a cuvette at 25°C. Rates of hydrolysis were measured as the rate of change of absorbance at 410nm, after a delay of 75 seconds. The assay was linear up to rates of 0.4 absorption units/min.

The amylase protein concentration was measured using the standard Bio-Rad assay (Bio-Rad Laboratories) based on the method of

Bradford, M. (1976) Anal. Biochem. 72:248) using bovine serum albumin standards.

Starch Hydrolysis Assay: The standard method for assaying the alpha-amylase activity of Spezyme® AA20 was used. This method is described in detail in Example 1 of USSN 07/785,624, incorporated herein by reference. Native starch forms a blue color with iodine but fails to do so when it is hydrolyzed into shorter dextrin molecules. The substrate is soluble Lintner starch 5gm/liter in phosphate buffer, pH 6.2 (42.5gm/liter potassium dihydrogen

phosphate, 3.16gm/liter sodium hydroxide). The sample is added in 25mM calcium chloride and activity is measured as the time taken to give a negative iodine test upon incubation at 30°C. Activity is recorded in liquefons per gram or ml (LU) calculated according to the formula:

Figure imgf000025_0001

Where LU=liquefon unit

V=volume of sample (5ml)

t=dextrinization time (minutes)

D=dilution factor=dilution volume/ml or g of added enzyme.

Figure imgf000025_0002

Example 4

Characterization of Vaariant 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 Na2CO3. 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.

Figure imgf000026_0001

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. licheniformis by cassette mutagenesis, as outlined in Fig. 12: 1) Site directed mutagenesis (via primer extension in M13) was used to introduce unique restriction sites flanking the M15 codon to facilitate insertion of a mutagenesis cassette. Specifically, a BstBI site at codons 11-13 and a Mscl site at codons 18-20 were introduced using the two oligonucleotides shown below:

Figure imgf000027_0001

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.

Figure imgf000028_0001

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 4. The results are shown in Fig. 16.

Example 7

Characterization of M197X Variants

As can be seen in Fig. 9, there was a wide range of amylase activity (measured in the soluble substrate assay) expressed by the M197X (A4 form) variants. The amylases were partially purified from the supernatants by precipitation with two volumes of ethanol and resuspension. They were then screened for thermal stability (Fig. 10) by heating at 95°C for 5 minutes in lOmM acetate buffer pH 5.0, in the presence of 5mM calcium chloride; the A4 wild-type retained 28% of its activity after incubation. For M197W and M197P we were unable to recover active protein from the supernatants. Upon sequencing, the M197H variant was found to contain a second

mutation, N190K. M197L was examined in a separate experiment and was one of the lowest thermally stable variants. There appears to be a broad correlation between expression of amylase activity and thermal stability. The licheniformis amylase is restricted in what residues it can accommodate at position 197 in terms of retaining or enhancing thermal stability: cysteine and threonine are preferred for maximal thermal stability under these conditions whereas alanine and isoleucine are of intermediate stability. However, other substitutions at position +197 result in lowered thermal stability which may be useful for other applications. Additionally, different substitutions at +197 may have other beneficial properties, such as altered pH performance profile or altered oxidative stability. For example, the M197C variant was found to inactivate readily by air oxidation but had enhanced thermal stability. Conversely, compared to the M197L variant, both M197T and M197A retained not only high thermal stability (Fig. 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 suJbtilis 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. licheniformis alpha-amylase with chloramine-T at pH 8.0 (AA20 = 0.65 mg/ml, M197A = 1.7 mg/ml, M197L = 1.7 mg/ml). The data shows that by changing the methionine at position 197 to leucine or alanine, the inactivation of 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 M197

Certain variants of W138 (F, Y and A) were made as double mutants, with M197T (made as per the disclosure of Example 3). The double mutants were made following the methods described in Examples 1 and 3. Generally, single negative strands of DNA were prepared from an M13MP18 clone of the 1.72kb coding sequence (Pst I-Sst I) of the B. licheniformis alpha-amylase M197T mutant. Site-directed

mutagenesis was done using the primers listed below, essentially by the method of Zoller, M. et al. (1983) except T4 gene 32 protein and T4 polymerase were substituted for klenow. The primers all

contained unique sites, as well as the desired mutation, in order to identify those clones with the appropriate mutation.

Figure imgf000032_0001

Mutants were identified by restriction analysis and W138F and W138Y confirmed by DNA sequencing. The W138A sequence revealed a

nucleotide deletion between the unique BspE I and Xma I sites, however, the rest of the gene sequenced correctly. The 1.37kb Sstll/Sstl fragment containing both W138X and M197T mutations was moved from M13MP18 into the expression vector pBLapr resulting in pBLapr (W138F, M197T) and pBLapr (W138Y, M197T). The fragment containing unique BspE I and Xma I sites was cloned into pBLapr (BspE I, Xma I, M197T) since it is useful for cloning cassettes containing other amino acid substitutions at position 138.

Single Mutations at Amino Acid Position 138

Following the general methods described in the prior examples, certain single variants of W138 (F, Y, L, H and C) were made. The 1.24kb Asp718-Sstl fragment containing the M197T mutation in plasmid pBLapr (W138X, M197T) of Example 7 was replaced by the wild- type fragment with methionine at 197, resulting in pBLapr (W138F), pBLapr (W138Y) and pBLapr (BspE I, Xma I).

The mutants W138L, W138H and W138C were made by ligating synthetic cassettes into the pBLapr (BspE I, Xma I) vector using the following primers:

Figure imgf000033_0001

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. licheniformis alpha-amylase should be modified for stability in a formulation comprising ADD + perborate + TAED.

Starch Hydrolysis Assay

A 4000 ppm solution of automatic dishwashing detergent (ADD) containing perborate and TAED was prepared in water with a hardness of 7 gpg and three cooked pieces of elbow macaroni were added. The amylase mutants described above were added to this ADD solution to yield a final concentration of 5 ppm active enzyme. The tubes were incubated at 50°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.

Figure imgf000035_0001

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.

Figure imgf000035_0002
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 (21-23ºC) 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 Stable Protease/Oxidatively Stable Amylase

Stability Studies

Enzyme granules containing either: 1) wild-type protease and wild- type amylase; or 2) bleach stable protease (GG36-M222S) made by the methods described in US Re 34606 and bleach stable amylase (AA20- M15T/W138Y/M197T) were dissolved in buffer containing 0.1 M sodium borate pH 10.2 and 0.005% Tween 80 at a concentration of 12.5 mg of each enzyme. To 9 ml of these solutions was added either 1 ml distilled water or 1 ml 30% hydrogen peroxide. After incubation of the solutions at 25°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.

Figure imgf000037_0001

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.

Figure imgf000038_0001
Figure imgf000039_0001

Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001

Figure imgf000045_0002

Figure imgf000046_0001
Figure imgf000046_0002
Figure imgf000046_0003
Figure imgf000047_0001
Figure imgf000047_0002
Figure imgf000047_0003
Figure imgf000048_0001
Figure imgf000049_0001

Figure imgf000049_0002
Figure imgf000050_0001
Figure imgf000050_0002
Figure imgf000050_0003
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000052_0002
Figure imgf000052_0003
Figure imgf000053_0001
Figure imgf000053_0002
Figure imgf000053_0003
Figure imgf000054_0001

Figure imgf000055_0001
Figure imgf000056_0001

Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001

Figure imgf000060_0001
Figure imgf000061_0001

Claims

WHAT IS CLAIMED IS :
1. An improved bleach-containing cleaning composition, the improvement comprising adding to the bleach-containing composition a mutant alpha-amylase that is the expression product of a mutated DNA sequence encoding an alpha-amylase, the mutated DNA sequence being derived from a precursor alpha-amylase by the substitution of a methionine at a position equivalent to M+197 in B. licheniformis alpha-amylase and the substitution of one or more methionine or tryptophan at a position equivalent to M+15 or W+138 in B.
licheniformis alpha-amylase.
2. An improved cleaning composition of Claim 1 wherein the cleaning composition is a dish care cleaning composition.
3. An improved cleaning composition of Claim 1 wherein the mutant alpha-amylase is selected from the group consisting of M15T/M197T; M15S/M197T; W138Y/M197T; M15S/W138Y/M197T and M15T/W138Y/M197T.
4. An improved cleaning composition of Claim 1 further comprising a mutant protease that is the expression product of a mutated DNA sequence encoding a protease, the mutated DNA sequence being derived from a precursor protease by the substitution of a methionine at a position equivalent to M+222 in Bacillus amyloliquefaciens protease.
5. An improved cleaning composition of Claim 4 wherein the mutant protease comprises a substitution selected from the group of amino acids consisting of alanine, cysteine and serine.
6. An improved cleaning composition of Claim 4 comprising an alpha-amylase mutant selected from the group consisting of
M15T/M197T, M15S/M197T, W138Y/M197T, M15S/W138Y/M197T and
M15T/W138Y/M197T, and a protease mutant selected from the group consisting of M222C, M222S and M222A.
7. An improved cleaning composition of Claim 6 which is a
granular composition.
PCT/US1995/010426 1994-08-11 1995-08-09 An improved cleaning composition WO1996005295A2 (en)

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US08/289,351 1994-08-11

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MX9700776A MX9700776A (en) 1994-08-11 1995-08-09 An improved cleaning composition.
AU33662/95A AU686007B2 (en) 1994-08-11 1995-08-09 An improved cleaning composition
NZ29198495A NZ291984A (en) 1994-08-11 1995-08-09 Bleach-containing cleaning composition comprising a mutant alpha-amylase and optionally a mutant protease
KR1019970700864A KR970704872A (en) 1994-08-11 1995-08-09 The improved cleaning compositions
BR9508582A BR9508582A (en) 1994-08-11 1995-08-09 cleansing composition containing bleaching agent
EP19950930186 EP0775201A2 (en) 1994-08-11 1995-08-09 An improved cleaning composition
JP50760396A JPH10504197A (en) 1994-08-11 1995-08-09 Improved cleaning compositions
FI970563A FI970563A (en) 1994-08-11 1997-02-10 Improved cleaning composition
NO970609A NO970609L (en) 1994-08-11 1997-02-10 Improved cleaning composition

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US5958739A (en) * 1996-06-06 1999-09-28 Genencor International Inc. Mutant α-amylase
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WO2008153805A2 (en) * 2007-05-30 2008-12-18 Danisco Us, Inc., Genencor Division Improved variants of the bacillus licheniformis alpha-amylase
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WO2016106011A1 (en) 2014-12-23 2016-06-30 E. I. Du Pont De Nemours And Company Enzymatically produced cellulose
WO2016201044A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc Osmotic burst encapsulates
WO2016201069A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc Low-density enzyme-containing particles
WO2016201040A1 (en) 2015-06-09 2016-12-15 Danisco Us Inc. Water-triggered enzyme suspension
WO2016205755A1 (en) 2015-06-17 2016-12-22 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
WO2017079751A1 (en) 2015-11-05 2017-05-11 Danisco Us Inc Paenibacillus sp. mannanases
WO2017079756A1 (en) 2015-11-05 2017-05-11 Danisco Us Inc Paenibacillus and bacillus spp. mannanases
WO2017083229A1 (en) 2015-11-13 2017-05-18 E. I. Du Pont De Nemours And Company Glucan fiber compositions for use in laundry care and fabric care
WO2017083228A1 (en) 2015-11-13 2017-05-18 E. I. Du Pont De Nemours And Company Glucan fiber compositions for use in laundry care and fabric care
WO2017083226A1 (en) 2015-11-13 2017-05-18 E. I. Du Pont De Nemours And Company Glucan fiber compositions for use in laundry care and fabric care
WO2017106676A1 (en) 2015-12-18 2017-06-22 Danisco Us Inc Polypeptides with endoglucanase activity and uses thereof
WO2017192692A1 (en) 2016-05-03 2017-11-09 Danisco Us Inc Protease variants and uses thereof
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WO2018085524A2 (en) 2016-11-07 2018-05-11 Danisco Us Inc Laundry detergent composition
WO2018118917A1 (en) 2016-12-21 2018-06-28 Danisco Us Inc. Protease variants and uses thereof
WO2018118950A1 (en) 2016-12-21 2018-06-28 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
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US10167458B2 (en) 2011-06-30 2019-01-01 Novozymes A/S Alpha-amylase variants
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US5958739A (en) * 1996-06-06 1999-09-28 Genencor International Inc. Mutant α-amylase
US6080568A (en) * 1997-08-19 2000-06-27 Genencor International, Inc. Mutant α-amylase comprising modification at residues corresponding to A210, H405 and/or T412 in Bacillus licheniformis
EP2261359A1 (en) 1998-06-10 2010-12-15 Novozymes A/S Mannanases
EP2287318A1 (en) 1998-06-10 2011-02-23 Novozymes A/S Mannanases
EP2284272A1 (en) 1998-06-10 2011-02-16 Novozymes A/S Mannanases
US7713723B1 (en) 2000-08-01 2010-05-11 Novozymes A/S Alpha-amylase mutants with altered properties
US9670471B2 (en) 2001-05-15 2017-06-06 Novozymes A/S Alpha-amylase variant with altered properties
US8252573B2 (en) 2001-05-15 2012-08-28 Novozymes A/S Alpha-amylase variant with altered properties
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US9080137B2 (en) 2001-05-15 2015-07-14 Novozymes A/S Alpha-amylase variant with altered properties
US8486681B2 (en) 2001-05-15 2013-07-16 Novozymes A/S Alpha-amylase variant with altered properties
US9796968B2 (en) 2001-05-15 2017-10-24 Novozymes A/S Alpha-amylase variant with altered properties
US8080401B2 (en) 2004-10-01 2011-12-20 Henkel Ag & Co. Kgaa Alpha-amylase variants having an elevated solvent stability, method for the production thereof and detergents and cleansers containing these alpha-amylase variants
DE102004047777B4 (en) 2004-10-01 2018-05-09 Basf Se Alpha-amylase variants having an elevated solvent stability, method for their preparation and their use
WO2008153805A3 (en) * 2007-05-30 2009-05-14 Wolfgang Aehle Improved variants of the bacillus licheniformis alpha-amylase
WO2008153805A2 (en) * 2007-05-30 2008-12-18 Danisco Us, Inc., Genencor Division Improved variants of the bacillus licheniformis alpha-amylase
US7618801B2 (en) 2007-10-30 2009-11-17 Danison US Inc. Streptomyces protease
US7879788B2 (en) 2007-10-30 2011-02-01 Danisco Us Inc. Methods of cleaning using a streptomyces 1AG3 serine protease
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CA2197203A1 (en) 1996-02-22
NO970609L (en) 1997-03-24
CO4440440A1 (en) 1997-05-07
AU686007B2 (en) 1998-01-29
FI970563A (en) 1997-02-10
MX9700776A (en) 1997-05-31
NZ291984A (en) 1998-04-27
EP0775201A2 (en) 1997-05-28
PL318209A1 (en) 1997-05-26
JPH10504197A (en) 1998-04-28
KR970704872A (en) 1997-09-06
NO970609D0 (en) 1997-02-10
HUT77748A (en) 1998-07-28
AU3366295A (en) 1996-03-07
WO1996005295A3 (en) 1996-03-28
FI970563A0 (en) 1997-02-10
FI970563D0 (en)
CN1158637A (en) 1997-09-03

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