# Patent

## Title
Halomonas strain WDG195-related alpha-amylases, and methods of use, thereof

## Abstract

Compositions and methods relating to an alpha-amylase enzyme obtained from Halomonas variabilis WDG195 are described.

## Background
PRIORITY
The present application claims priority under 35 USC §371 to International Application No. PCT/US2010/025368, filed Feb. 25, 2010, which claims the benefit of to U.S. Provisional Patent Application Ser. No. 61/167,612, filed on Apr. 8, 2009, both of which are hereby incorporated by reference in their entirety.


SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “31341US_SequenceListing”, created on Mar. 19, 2012, which is 48,605 bytes in size.
TECHNICAL FIELD
Disclosed are compositions and methods relating to α-amylase enzymes obtained from Halomonas strain WDG195, and to structurally related amylases.
BACKGROUND
Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose consists of linear chains of α-1,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000. Amylopectin is a branched polymer containing α-1,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
Sugars from starch, in the form of concentrated dextrose syrups, are currently produced by an enzyme catalyzed process involving: (1) liquefaction (or viscosity reduction) of solid starch with an α-amylase into dextrins having an average degree of polymerization of about 7-10, and (2) saccharification of the resulting liquefied starch (i.e. starch hydrolysate) with amyloglucosidase (also called glucoamylase or GA). The resulting syrup has a high glucose content. Much of the glucose syrup that is commercially produced is subsequently enzymatically isomerized to a dextrose/fructose mixture known as isosyrup.
α-amylases (EC 3.2.1.1) hydrolyze starch, glycogen, and related polysaccharides by cleaving internal α-1,4-glucosidic bonds at random. For a number of years, α-amylase enzymes have been used for a variety of different purposes, including starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing. These enzymes can also be used to remove starchy stains during dishwashing and laundry washing. Laundry and dish soils vary greatly in composition and therefore also in their ability to be removed and few amylases in the market place can be used for both laundry and dish applications.
Halomonas species are Gram-negative, moderately halophilic bacteria that grow optimally in media containing 3-15% NaCl, although most will grow over a very wide range of salinities. Some species are alkaliphilic. The only described amylase from Halomonas sp. is from Halomonas meridiana DSM5425 (Coronado, M. J. et al. (2000) FEMS Microbiology Letters 183:67-71). This α-amylase (i.e., amyH) has been cloned and a deduced amino acid sequence (AmyH) predicted (Coronado, M. J. et al., (2000) Microbiology 146:861-868). The nucleotide sequence for the amyH gene (AJ239061) and the predicted amino acid sequence for the AmyH polypeptide (CAB92963) are available in GenBank. The amino acid sequence of AmyH is also set forth in SEQ ID NO: 1. AmyH is predicted to be a polypeptide of 457 amino acids, including a signal sequence of 20 amino acids and a mature polypeptide sequence of 437 amino acids.
While AmyH appears to belong to be a family 13 glycosyl hydrolases, it has low sequence identity with its closest homologues, as shown in the following Table:












Sequence


Homolog
identity







α-amylase, catalytic region (Herpetosiphon aurantiacus
54%


ATCC 23779)


α-amylase (Stigmatella aurantiaca DW4/3-1)
54%


Glycosidase (Pseudoalteromonas tunicata D2)
54%


α-Amylase from Pseudoalteromonas haloplanctis
52%









SUMMARY
The present compositions and methods relate to an α-amylase from Halomonas strain WDG195 and related α-amylases, which represent a unique family of amylases useful for industrial applications. The amylases are referred to collectively as AmyWDG195-related polypeptides or AmyWDG195-related amylases.
In one aspect, an isolated polypeptide is provided, having at least 80% amino acid sequence identity to AmyWDG195 and including at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195, as determined by aligning the amino acid sequence of the polypeptide with the amino acid sequence of AmyWDG195: E at position 3, E at position 20, M at position 25, N at position 26, L at position 40, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, L at position 267, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, L at position 307, D at position 340, V at position 350, S at position 360, L at position 381, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479.
In some embodiments, the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: E at position 3, E at position 20, M at position 25, N at position 26, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, D at position 340, V at position 350, S at position 360, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479. As above, the AmyWDG195-related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
In some embodiments, the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: M at position 25, N at position 26, L at position 177, H at position 179, E at position 294, T at position 298, and M at position 449.
In some embodiments, the polypeptide includes at least one of the following combinations of amino acid residues at the indicated amino acid position relative to AmyWDG195: M at position 25 and N at position 26, optionally with E at position 20; Q at position 91 and G at position 94; L at position 177, H at position 179, and G at position 181; D at position 280 and V at position 281, optionally with D at position 278; R at position 297 and T at position 298, optionally with E at position 294; E at position 386 and D at position 390, optionally with L at position 381; and M at position 449 and H at position 453.
In some embodiments, the polypeptide includes at least 30 glutamate residues.
In some embodiments, the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: N at position 104, D at position 194, N at position 200, and H at position 235.
In some embodiments, the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: Y at position 302, G at position 300, N at position 427, D at position 404, and W at position 403
In some embodiments, the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: N at position 289, V at position 324, D at position 325, and S at position 337.
In some embodiments, the polypeptide includes the following amino acid residue at the indicated amino acid position relative to AmyWDG195: L at position 197.
In some embodiments, AmyWDG195 has the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polypeptide has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polypeptide has at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 12. In particular embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 12 (AmyWDG195).
In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 23 (AmySWT282).
In some embodiments, the polypeptide has α-amylase activity. In some embodiments, the activity is independent of calcium.
In another aspect, a cultured cell material comprising one or more of any of the aforementioned polypeptides is provided.
In another aspect, a composition comprising one or more of any of the aforementioned polypeptides is provided. In some embodiments, the composition is a cleaning composition. In some embodiments, the composition is effective for removing starchy stains from laundry, dishes, or textiles.
In another aspect, a method for removing a starchy stain from a surface is provided, comprising
incubating the surface in the presence of a aqueous composition comprising an effective amount of a polypeptide of claim 1,
allowing the polypeptides to hydrolyse starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition,
thereby removing the starchy stain from the surface.
In some embodiments, the incubating is in the absence of calcium. In some embodiments, the incubating is in the presence of a calcium chelating agent. In some embodiments, the surface is a textile surface.
In another aspect, a method for expressing a polypeptide having amylase activity is provided, comprising:
constructing an expression vector comprising a polynucleotide encoding a signal sequence linked to a polynucleotide encoding a polypeptide of claim 1;
introducing the expression vector into a host cell,
expressing the polypeptides in the presence of at least about 1% NaCl,
recovering the polypeptide expressed.
In some embodiments, expressing the polypeptide is performed in the presence of at least about 2% NaCl. In some embodiments, expressing the polypeptide is performed in the presence of at least about 3% NaCl.
In some embodiments, the amount of polypeptide expressed in the presence of 1% NaCl is at least 5-fold the amount expressed in minimal media without added NaCl. In some embodiments, the amount of polypeptide expressed in the presence of 2% NaCl is at least 20-fold the amount expressed in minimal media without added NaCl. In some embodiments, the amount of polypeptide expressed in the presence of 1% NaCl is at least 20-fold the amount expressed in minimal media without added NaCl.
In a further aspect, a vector comprising a polynucleotide sequence encoding an AmyWDG195-related amylase is provided. The AmyWDG195-related amylase coding sequence may be operably linked to a promoter or to other control elements. In yet a further aspect, a host cell comprising a polynucleotide encoding an AmyWDG195-related amylase, or vector comprising such a polynucleotide, is provided. The isolated host cell can be a prokaryote or eukaryote. The isolated host cell can be a bacterium (e.g., B. subtilis, B. licheniformis, B. lentus, B. brevis, Geobacillus (formerly Bacillus) stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. thuringiensis, Streptomyces lividans, S. murinus, or Escherichia coli).
Another aspect contemplates a detergent additive comprising an AmyWDG195-related amylase, wherein the detergent additive is optionally in the form of a non-dusting granulate, microgranulate, stabilized liquid, gel, or protected enzyme. The polypeptide in the detergent additive can be a truncated polypeptide as described above. The detergent additive can contain about 0.02 mg to about 200 mg of polypeptide per gram of the detergent additive. The detergent additive can further comprise an enzyme selected from the group consisting of a protease, a lipase, a peroxidase, an oxidase, an amylolytic enzyme, a cellulase, a polyesterase, and any combination thereof.
Another aspect contemplates a detergent composition comprising any of the described detergent additives. A detergent composition can optionally comprise one or more of: a surfactant, a bleaching system or bleach, a detergent builder, a polymer, a stabilizing agent, a fabric conditioner, a foam booster, a suds suppressor, an anti-corrosion agent, a dye, a perfume, a soil suspending agent, a tarnish inhibitor, an optical brightener, or a bacteriocide. A detergent composition can comprise or further comprise an additional enzyme, wherein the enzyme is a protease, a lipase, a peroxidase, an oxidase, an amylolytic enzyme, a cellulase, a polyesterase, or any combination thereof.
Another aspect contemplates a manual or automatic dishwashing detergent composition comprising an AmyWDG195-related amylase.
Yet a further aspect contemplates a method of washing dishes comprising applying a manual or automatic dishwashing detergent described herein to a dish or dishes in need thereof. The method of washing the dishes contemplates adding the dishwashing detergent in an amount such that the wash liquor contains a polypeptide described herein in the amount of about 0.01 ppm to about 4 ppm.
Another aspect contemplates a laundry detergent composition comprising a detergent additive described herein. Yet a further aspect contemplates a method of cleaning a textile comprising washing a soiled textile in solution with a detergent composition described herein. The method further contemplates having the polypeptide described herein in an amount in the solution of about 0.01 to about 2 ppm in the solution.
These and other aspects and embodiments of the compositions and methods will be apparent from the present description and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a diagram of the vector pRANGER-BTB3-Cat.
FIG. 2 shows the results of a BLAST search showing that AmyWDG195 belongs to the α-amylase superfamily.
FIG. 3 shows a diagram of the expression vector pHPLT.
FIGS. 4 and 5 show the results of assays to evaluate cleaning performance.
FIG. 6 is an image of a Coomassie-stained gel showing expression of AmySWT282.
FIG. 7 shows and alignment of AmyWDG195, AmySWT282, and Amy K38, emphasizing residues conserved between AmyWDG195 and AmySWT282.
FIG. 8 shows and alignment of AmyWDG195, AmySWT282, and Amy K38, showing conserved residues and a consensus sequence.
FIG. 9 lists the polypeptide and nucleotide sequences referred to in the description.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 is the amino acid sequence of the AmyH precursor (i.e., immature or full-length) polypeptide from Halomonas meridiana, The signal sequence is the first about 20 residues.
SEQ ID NO: 2 is a partial nucleotide sequence of the 16S rRNA gene of Halomonas strain WDG195
SEQ ID NOs: 3-8 are the nucleotide sequences of primers described in the Examples.
SEQ ID NO: 9 is the nucleotide sequence of the amyWDG195 gene.
SEQ ID NO: 10 is the amino acid sequence of the AmyWDG195 precursor (i.e., immature or full-length) polypeptide. The signal sequence is the first about 22 residues, with the remaining residue representing the mature polypeptide.
SEQ ID NO: 11 is the amino acid sequence of the AmyWDG195 signal peptide.
SEQ ID NO: 12 is the amino acid sequence of the AmyWDG195 mature polypeptide.
SEQ ID NOs: 13-17 are the nucleotide sequences of primers described in the Examples.
SEQ ID NO: 18 is the partial nucleotide sequence of the AmySWT282 gene.
SEQ ID NO: 19 is the nucleotide sequence encoding the AmySWT282 precursor (i.e., immature or full-length) polypeptide.
SEQ ID NO: 20 is the nucleotide sequence encoding the AmySWT282 mature polypeptide.
SEQ ID NO: 21 is nucleotide sequence encoding the AmySWT282 signal peptide.
SEQ ID NO: 22 is the amino acid sequence of the AmySWT282 precursor (i.e., immature or full-length) polypeptide. The signal sequence is the first about 22 residues, with the remaining residue representing the mature polypeptide.
SEQ ID NO: 23 is the amino acid sequence of the AmySWT282 mature polypeptide.
SEQ ID NO: 24 is the amino acid sequence of the AmySWT282 signal peptide.
SEQ ID NOs: 25-33 are the nucleotide sequences of primers described in the Examples.
SEQ ID NO: 34 is the amino acid sequence of the AmyK38 precursor (i.e., immature or full-length) polypeptide (gi16874476). The signal sequence is the first about 21 residues, with the remaining residue representing the mature polypeptide SEQ ID NO: 35 is the amino acid sequence of the AmyK38 mature polypeptide.
SEQ ID NOs: 36-41 are the nucleotide sequences of primers described in the Examples.
DETAILED DESCRIPTION
Described are compositions and methods relating to an α-amylase isolated from Halomonas strain WDG195, herein called “AmyWDG195” (SEQ ID NO: 12). While this amylase belongs to the α-amylase super-family, it is unrelated to AmyH (SEQ ID NO: 1), the amylase previously isolated from Halomonas meridiana strain DSM5425, which is assigned to the family 13 glycosyl hydrolases. In particular, AmyWDG195 and AmyH share less than 11% sequence identity.
AmyWDG195 shares significant sequence identity with another heretofore unknown amylase from Bacillus sp. strain SWT282, herein called “AmySWT282” (SEQ ID NO: 23), and to a known amylase from Bacillus sp. strain K38, herein called “AmyK38” (SEQ ID NO: 35; GenBank accession number BAB71820; Hagihara, H. et al. (2001) Appl. Environ. Microbiol. 67:1744-50). In particular, AmyWDG195 shares about 78.1% sequence identity with AmySWT282 and 81.5% sequence identity with AmyK38. Notably, AmySWT282 and AmyK38 share 77.9% sequence identity; therefore, these three amylases are approximately equidistant in evolutionary distance. However, a more refined structural analysis reveals that while AmyWDG195, AmySWT282, AmyK38, share some common features, AmyWDG195 and AmySWT282 are readily distinguishable from AmyK38 based on other features.
In particular, both AmyWDG195 and AmySWT282 include key residues that are associated with binding to three sodium ions in AmyK38 (Hagihari, H. et al. (2001) Eur. J. Biochem. 268:3974-82; Nonaka, T. et al. (2003) J. Biol. Chem. 278:24818-25). In particular, the presence of N at position 104, D at position 194, N at position 200, and H at position 235, which is associated with the binding of AmyK38 to a first sodium, is conserved in both AmyWDG195 and AmySWT282. Similarly, the presence of Y at position 302, G at position 300, N at position 427, D at position 404, and W at position 403, which is associated with the binding of AmyK38 to a second sodium, and the presence of N at position 289, V at position 324, D at position 325, and S at position 337, which is associated with the binding of AmyK38 to a third sodium, is conserved in both AmyWDG195 and AmySWT282. Accordingly, AmyWDG195 and AmySWT282 are expected to share certain halophilic properties with AmyK38, which is confirmed by experiments described herein. In addition, AmyWDG195, AmySWT282, and AmyK38 all share an L at position 197, unlike many other Bacillus amylases which have an M at this position. This makes these amylases less susceptible to chemical oxidation than other Bacillus amylases.
In contrast, AmyWDG195 and AmySWT282 can readily be distinguished from AmyK38 based on amino acid residues present at each of 32 different positions, at which AmyWDG195 and AmySWT282 share the same amino acid residue, which is different from that present in AmyK38. AmyWDG195 and AmySWT282 also contain a significantly larger number of glutamate residues and have lower predicted isoelectric points (pI) than AmyK38. Thus, AmyWDG195 and AmySWT282, represent related, heretofore unknown, calcium-independent amylases, which are resistant to chelating reagents and chemical oxidants, making them very attractive for cleaning applications.
For the purposes of the present description, AmyWDG195, AmySWT282, and related polypeptides are referred to collectively as AmyWDG195-related polypeptides or AmyWDG195-related amylases. For convenience, the amino acid position numbering of AmyWDG195 and AmyK38 is used with reference to these polypeptides (see, e.g., FIGS. 7 and 8). However, AmySWT28 is one amino acid shorter at the N-terminus than AmyWDG195 and AmyK38, thereof the actual amino acid positions of AmySWT28 are one less (i.e., −1) with respect to AmyWDG195 and AmyK38.
Various compositions and methods that involve AmyWDG195-related amylase polypeptides, or polynucleotides encoding these polypeptides, are now to be described.
1. Definitions and Acronyms
In accordance with this detailed description, the following abbreviations and definitions apply. Note that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an enzyme” includes a plurality of such enzymes, and reference to “the dosage” includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following abbreviations and/or terms are defined for clarity:
1.1 Abbreviations/Acronyms
The following abbreviations/acronyms have the following meanings unless otherwise specified:

    
    
        AE alcohol ethoxylate
        AEO alcohol ethoxylate
        AEOS alcohol ethoxysulfate
        AES alcohol ethoxysulfate
        AmyWDG195 α-amylase from Halomonas strain WDG195
        AmyH α-amylase from Halomonas meridiana strain DSM5425
        AmySWT282 α-amylase from Bacillus sp. strain SWT282
        AmyK38 α-amylase from Bacillus sp. strain K38
        AOS α-olefinsulfonate
        AS alkyl sulfate
        CBD-25 carbohydrate binding domain protein family 25
        cDNA complementary DNA
        CMC carboxymethylcellulose
        DNA deoxyribonucleic acid
        DTMPA diethylenetriaminepentaacetic acid
        EC enzyme commission
        EDTA ethylenediaminetetraacetic acid
        EMPA Eidgenössische Materialprüfungs- und Forschungs Anstalt (Swiss Federal Laboratories for Materials Testing and Research)
        EO ethylene oxide (polymer fragment)
        F&HC fabric & household care
        GA glucoamylase
        IPTG isopropyl β-D-thiogalactoside
        kDa kiloDalton
        LAS linear alkylbenzenesulfonate
        LAT pertaining to B. licheniformis amylase
        MW molecular weight
        MWU modified Wohlgemuth unit; 1.6×10−5 mg/MWU=unit of activity
        NOBS nonanoyloxybenzenesulfonate
        NTA nitriloacetic acid
        OxAm Purastar HPAM 5000L (Genencor International, Inc.)
        PEG polyethyleneglycol
        pI isoelectric point
        PVA poly(vinyl alcohol)
        PVP poly(vinylpyrrolidone)
        RNA ribonucleic acid
        SAS alkanesulfonate
        SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
        sp. species
        TAED tetraacetylethylenediamine
        w/v weight/volume
        w/w weight/weight
        v/v volume/volume
        wt % weight percent
        ° C. degrees Centigrade
        H2O water
        dH2O or DI deionized water
        dIH2O deionized water, Milli-Q filtration
        g or gm gram
        μg microgram
        mg milligram
        kg kilogram
        μL and μl microliter
        mL and ml milliliter
        mm millimeter
        μm micrometer
        M molar
        mM millimolar
        μM micromolar
        U unit
        sec and ′′ second
        min and ′ minute
        hr hour
        DO dissolved oxygen
        Genencor Danisco US Inc, Genencor Division, Palo Alto, Calif.
        Nem Newton centimeter
        ETOH ethanol
        eq. equivalent
        N normal
        ds or DS dry solids content
    
    


1.2 Definitions
The terms “amylase” or “amylolytic enzyme” refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch. Amylases are hydrolases that cleave the α-D-(1→4) O-glycosidic linkages in starch. Generally, α-amylases (EC 3.2.1.1; α-D-(1→4)-glucan glucanohydrolase) are defined as endo-acting enzymes cleaving α-D-(1→4) O-glycosidic linkages within the starch molecule in a random fashion. In contrast, the exo-acting amylolytic enzymes, such as β-amylases (EC 3.2.1.2; α-D-(1→4)-glucan maltohydrolase) and some product-specific amylases like maltogenic α-amylase (EC 3.2.1.133) cleave the starch molecule from the non-reducing end of the substrate. β-Amylases, α-glucosidases (EC 3.2.1.20; α-D-glucoside glucohydrolase), glucoamylase (EC 3.2.1.3; α-D-(1→4)-glucan glucohydrolase), and product-specific amylases can produce malto-oligosaccharides of a specific length from starch.
As used herein the term “starch” refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C6H10O5)x, wherein X can be any number. The term includes plant-based materials such as grains, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassaya, millet, potato, sweet potato, and tapioca.
The terms, “wild-type,” “parental,” or “reference,” with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms “wild-type,” “parental,” or “reference,” with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.
The term “variant,” with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes a man-made substitution, insertion, or deletion at one or more amino acid positions. Similarly, a the term “variant,” with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.
The term “recombinant,” when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
The terms “recovered,” “isolated,” and “separated,” refer to a compound, protein (polypeptides), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature.
As used herein, the term “purified” refers to material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.
The terms “thermostable” and “thermostability,” with reference to an enzyme, refer to the ability of the enzyme to retain activity after exposure to an elevated temperature. The thermostability of an enzyme, such as an amylase enzyme, is measured by its half-life (t1/2) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residual α-amylase activity following exposure to (i.e., challenge by) an elevated temperature.
A “pH range,” with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.
As used herein, the terms “pH stable” and “pH stability,” with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min, 1 hour).
As used herein, the term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N→C).
The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences which encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5′-to-3′ orientation.
By “homologue” shall mean an entity having a specified degree of identity with the subject amino acid sequences and the subject nucleotide sequences. A homologous sequence is taken to include an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identical to the subject sequence, using conventional sequence alignment tools (e.g., Clustal, BLAST, and the like). Typically, homologues will include the same active site residues as the subject amino acid sequence, unless otherwise specified.
As used herein, “hybridization” refers to the process by which one strand of nucleic acid base pairs with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by the following: 50° C. and 0.2×SSC (1×SSC=0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0). Highly stringent conditions are exemplified by the following: 65° C. and 0.1×SSC (1×SSC=0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0)].
As used herein, a “synthetic” molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.
As used herein, the terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, “transformation” or “transduction,” as known in the art.
A “host strain” or “host cell” is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., an AmyWDG195-related polypeptide) has been introduced. Exemplary host strains are bacterial cells. The term “host cell” includes protoplasts created from cells, such as those of a Bacillus sp.
The term “heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.
The term “endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
As used herein, the term “expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.
A “selective marker” or “selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.
A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
The term “operably linked” means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.
A “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.
As used herein, “biologically active” refer to a sequence having a specified biological activity, such an enzymatic activity.
“Water hardness” is a measure of the minerals (e.g., calcium and magnesium) present in water.
As used herein, “a cultured cell material comprising an AmyWDG195-related amylase,” or similar language, refers to a cell lysate or supernatant that includes an AmyWDG195-related amylase as a component. The cell material is preferably from a heterologous host that is grown in culture for the purpose of producing the AmyWDG195-related amylase.
All reference cited, herein, are hereby incorporated by reference in their entirety.
2. AmyWDG195-Related Amylase Nucleic Acids and Polypeptides
One aspect of the present compositions and methods is an AmyWDG195-related polypeptide. The polypeptide may correspond to AmyWDG195, AmySWT282, an amylase having a specified degree of identity to AmyWDG195 or AmySWT282, variants of AmyWDG195 or AmySWT282 that include man-made substitutions, insertions, or deletions, or chimeras, thereof. Exemplary AmyWDG195 and AmySWT282 polypeptides have the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 23, respectively. Additional polypeptides have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% homology/identity to an AmyWDG195 or AmySWT282 polypeptide, such as the polypeptides of SEQ ID NO: 12 and SEQ ID NO: 23. The present compositions and methods exclude an Amy K38 polypeptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 35.
As noted above, AmyWDG195 and AmySWT282 can be distinguished from AmyK38 based on the amino acid residue that is present at each of 32 different positions, at which AmyWDG195 and AmySWT282 share the same amino acid residue, which is different from that present in AmyK38. Positions occupied by the same amino acid residues in AmyWDG195 and AmySWT282 (using AmyWDG195 numbering) but different in Amy K38 are summarized in the following Table:














Common positions




in AmyWDG195
Equivalent positions



and AmySWT282
in AmyK38









Glu3
Leu3



Glu20
Gln20



Met25
Leu25



Asn26
His26



Leu40
Ile40



Asp68
Asn68



Glu82
Gln82



Gln91
Lys91



Gly94
Asp94



Leu177
Phe177



His179
Asn179



Gly182
Asn182



Asn225
Asp225



Leu267
Val267



Asp278
Asn278



Asp280
Glu280



Val281
Met281



Glu294
Arg294



Arg297
Gln297



Thr298
Gln298



Leu307
Ile307



Asp340
Ala340



Val350
Thr350



Ser360
Asn360



Leu381
Ile381



Glu386
Asp386



Asp390
Asn390



Gly418
Asn418



Met449
Leu449



His453
Asn453



Asp470
Asn470



Gln479
Asn479










Referring to the above Table and the alignment shown in FIG. 7, in some embodiments, the AmyWDG195-related polypeptide has one of more of the following amino acid residues at the indicated positions: E at position 3, E at position 20, M at position 25, N at position 26, L at position 40, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, L at position 267, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, L at position 307, D at position 340, V at position 350, S at position 360, L at position 381, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479. The AmyWDG195-related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
In some embodiments, the AmyWDG195-related polypeptide has one of more of the following amino acid residues: E at position 3, E at position 20, M at position 25, N at position 26, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, D at position 340, V at position 350, S at position 360, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479. As above, the AmyWDG195-related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
In some embodiments, the AmyWDG195-related polypeptide has one of more of the following amino acid residues: M at position 25, N at position 26, L at position 177, H at position 179, E at position 294, T at position 298, and M at position 449. Again, the AmyWDG195-related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
In some embodiments, the AmyWDG195-related polypeptide has one of more of the following combinations of amino acid residues at the indicated positions: M at position 25 and N at position 26, optionally with E at position 20; Q at position 91 and G at position 94; L at position 177, H at position 179, and G at position 181; D at position 280 and V at position 281, optionally with D at position 278; R at position 297 and T at position 298, optionally with E at position 294; E at position 386 and D at position 390, optionally with L at position 381; and M at position 449 and H at position 453. The AmyWDG195-related polypeptide may have any subset of these amino acid residue combination, or all of these amino acid residue combinations.
In some embodiments, the AmyWDG195-related polypeptide retains N at position 104, D at position 194, N at position 200, and/or H at position 235, which is believed to be associated with binding to a first sodium, Y at position 302, G at position 300, N at position 427, D at position 404, and/or W at position 403, which is believed to be associated with binding to a second sodium, and/or N at position 289, V at position 324, D at position 325, and S at position 337, which is believed to be associated with binding to a third sodium. In some embodiments, the AmyWDG195-related polypeptide retains L at position 197, believed to associated with reduced susceptibility to chemical oxidation.
Also as noted above, AmyWDG195 and AmySWT282 contain a significantly larger number of glutamate residues and have lower predicted isoelectric points (Pi) than AmyK38. These differences are summarized in the following Table:















Amylase
Number of glutamates
pI









AmyWDG195
36
4.42



AmySWT282
33
4.47



AmyK38
23
4.73










Thus, in some embodiments, the AmyWDG195-related polypeptide has at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, or even at least 36 glutamate residues. In some embodiments, the AmyWDG195-related polypeptide has a pI of less than 4.7, less than 4.6, or even less than 4.5.
Where the AmyWDG195-related polypeptide is a variant having a specified degree of homology to AmyWDG195 or AmySWT282, one or more of the specified amino acid positions (or combinations, thereof) may be fixed, such that they do not change (i.e., are conserved or retained) in the variant, while the remaining residues are subject to variation. Alternatively or additionally, the number of glutamate residues in the polypeptide is maintained.
The polypeptides may be a “full-length” AmyWDG195-related amylase, which includes a signal sequence, or the mature form of an AmyWDG195-related amylase, which lacks the signal sequence. Exemplary immature forms of the AmyWDG195 and AmySWT282 polypeptides have the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 22, respectively, while exemplary mature forms of the AmyWDG195 and AmySWT282 polypeptides have the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 23, respectively. Mature forms of the polypeptides are most useful for use in cleaning compositions. The polypeptides may also be a truncated form of an AmyWDG195-related amylase, which lacks the N or C-terminus of the mature form, or fragments of an AmyWDG195-related amylase that retain at least a portion of the α-amylase activity characteristic of the parental AmyWDG195-related amylase.
As noted above, the polypeptides include variant polypeptides, such as those that include man-made substitutions. Exemplary substitutions are conservative amino acid substitutions, such as those listed in the following table.












For Amino Acid
Code
Replace with any of







Alanine
A
D-Ala, Gly, beta-Ala, L-Cys, D-Cys


Arginine
R
D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg,




Met, Ile, D-Met, D-Ile, Orn, D-Orn


Asparagine
N
D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln


Aspartic Acid
D
D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln


Cysteine
C
D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr


Glutamine
Q
D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp


Glutamic Acid
E
D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln


Glycine
G
Ala, D-Ala, Pro, D-Pro, b-Ala, Acp


Isoleucine
I
D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met


Leucine
L
D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met


Lysine
K
D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg,




Met, D-Met, Ile, D-Ile, Orn, D-Orn


Methionine
M
D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu,




Val, D-Val


Phenylalanine
F
D-Phe, Tyr, D-Thr, L-Dopa, His, D-His,




Trp, D-Trp, Trans-3,4, or 5-phenylproline,




cis-3,4, or 5-phenylproline


Proline
P
D-Pro, L-I-thioazolidine-4-carboxylic acid,




D-or L-1-oxazolidine-4-carboxylic acid


Serine
S
D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met,




Met(O), D-Met(O), L-Cys, D-Cys


Threonine
T
D-Thr, Ser, D-Ser, allo-Thr, Met,




D-Met, Met(O), D-Met(O), Val, D-Val


Tyrosine
Y
D-Tyr, Phe, D-Phe, L-Dopa, His, D-His


Valine
V
D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met









Preferred polypeptides retain α-amylase activity but may have altered biochemical properties with reference to a naturally-occurring parental polypeptide. In some cases, the parental polypeptide is that of SEQ ID NOs: 12 or 23.
The polypeptide may also be a chimeric polypeptide that includes at least a portion of an AmyWDG195-related amylase as described, and at least a portion of a second polypeptide. The second polypeptide may be, for example, a second amylase, a heterologous signal sequence, an epitope to allow tracking or purification, or the like. Exemplary heterologous signal sequences are from B. subtilis amylase (AmyE) or AprE, and Streptomyces CelA.
Another aspect of the present compositions and methods is a nucleic acid encoding an AmyWDG195-related polypeptide. The nucleic acid may encode AmyWDG195, AmySWT282, an amylase having a specified degree of identity to AmyWDG195 or AmySWT282, variants of AmyWDG195 or AmySWT282 that include man-made substitutions, insertions, or deletions, or chimeras, thereof. In one example, the nucleic acid encodes an amylase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% homology/identity to an AmyWDG195 or AmySWT282 polypeptide, such as the polypeptides of SEQ ID NO: 12 and SEQ ID NO: 20, respectively. Due to the degeneracy of the genetic code, it is understood that a plurality of nucleic acids may encode the same polypeptide.
The nucleic acid may also have a specified degree of homology to an exemplary polynucleotide encoding an AmyWDG195-related amylase, such as SEQ ID NO: 9, which encodes AmyWDG195 or SEQ ID NO: 20, which encodes AmySWT282. In one example, the nucleic acid has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity to one or both of these exemplary sequences. In another example, the nucleic acid hybridizes under stringent or very stringent conditions to one of these exemplary sequences.
Nucleic acids may encode a “full-length” or “fl” AmyWDG195-related amylase, which includes a signal sequence, only the mature form of an AmyWDG195-related amylase, which lacks the signal sequence, a truncated form of AmyWDG195-related amylase, which lacks the N or C-terminus of the mature form, or fragments, thereof that retain at least a portion of the α-amylase activity characteristic of the AmyWDG195-related amylase.
A nucleic acid that encodes an AmyWDG195-related amylase can be operably linked to various promoters and regulators in a vector for expression in various host cells. A nucleic acid that encodes an AmyWDG195-related amylase can also be linked to other coding sequences, e.g., to encode a chimeric polypeptide. Exemplary promoters are the B. subtilis Amy E and AprE promoters, and the Streptomyces CelA promoters.
3. Method of Producing and Purifying Proteins
Methods of producing and purifying proteins that are secreted in to the culture medium from Bacillus are known in the art, as are suitable host cells for producing α-amylases. Exemplary methods for producing the α-amylases are disclosed below.
3.1 Materials and Methods for Producing α-Amylases
An AmyWDG195-related amylase can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a suitable promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes. A large number of vectors are commercially available for use with recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, mini-chromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into an isolated host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. The integrated gene may also be amplified to create multiple copies of the gene in the chromosome by use of an amplifiable construct driven by antibiotic selection or other selective pressure, such as an essential regulatory gene or by complementation through dose effect of an essential metabolic pathway gene.
In the vector, the DNA sequence should be operably linked to a suitable promoter sequence. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding an AmyWDG195-related amylase, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis α-amylase gene (amyL), the promoters of the Geobacillus (formerly Bacillus) stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens α-amylase (amyQ), the promoters of the Bacillus subtilis xy1A and xy1B genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an AmyWDG195-related amylase polypeptide is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. For expression in Trichoderma reesei, the CBHII (cellobiohydrolase II) promoter may be used.
An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding an AmyWDG195-related amylase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ702.
The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xxsC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91/17243.
While intracellular expression or solid-state fermentation may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells, one aspect contemplates expression of an AmyWDG195-related amylase into the culture medium. In general, the α-amylase comprises a signal sequence at the amino terminus that permits secretion into the culture medium. If desirable, this signal peptide may be replaced by a different sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective signal polypeptide.
The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the AmyWDG195-related polypeptide to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the AmyWDG195-related polypeptide is operably linked to the control sequences in proper manner with respect to expression. A portion of an exemplary vector is depicted in FIG. 3.
The procedures used to ligate the DNA construct encoding an AmyWDG195-related polypeptide, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (see e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor, 1989, and 3rd ed., 2001).
An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an AmyWDG195-related amylase. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis; Lactobacillus spp. including Lactobacillus reuteri; Leuconostoc spp.; Pediococcus spp.; and Streptococcus spp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.
A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma reesei can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023.
In a yet further aspect, a method of producing an AmyWDG195-related amylase is provided comprising cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.
The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an AmyWDG195-related amylase. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
In one aspect, an enzyme secreted from the host cells is used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an alpha-amylase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the amylase to be expressed or isolated. The term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
An aspect contemplates the polynucleotide in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.
Host cells may be cultured under suitable conditions that allow expression of an AmyWDG195-related amylase. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sopharose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TnTTM (Promega) rabbit reticulocyte system.
An AmyWDG195-related amylase-expressing host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25° C. to about 75° C. (e.g., 30° C. to 45° C.), depending on the needs of the host and production of the desired AmyWDG195-related polypeptide. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 5.5 to about 8.0, again depending on the culture conditions needed for the host relative to production of an AmyWDG195-related amylase.
In some embodiments, an AmyWDG195-related amylase is expressed in the presence of additional NaCl added to the media, as compared to, for example, minimal media. The minimal media used herein included about 50 mM NaCl in addition to a small amount contributed by other component. The addition of 1%, 2%, or even 3% salt resulted in a significant increase in the amount of AmyWDG195-related amylase produced. Previous studies have suggested that NaCl was important for AmyK38 activity but not expression.
An aspect of the composition and method is the expression of an AmyWDG195-related polypeptide in the presence of additional NaCl, which increases the yield of polypeptide that is expressed in host cells. In some embodiments, the amount of NaCl is about 1% and the increase in yield is at least 3, at least 6, and even at least 9-fold. In some embodiments, the amount of NaCl is about 2% and the increase in yield is at least 4, at least 8, at least 12, and even at least 16-fold. In some embodiments, the amount of NaCl is about 3% and the increase in yield is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, and even at least 35-fold.
Generally, according to the method, an expression vector containing a polynucleotide encoding a signal sequence linked to a polynucleotide encoding an AmyWDG195-related polypeptide is constructed and introduced into a host cell. The AmyWDG195-related polypeptide is then expressed in host cells using medium enriched for NaCl as described, wherein the signal peptide directs secretion of the AmyWDG195-related polypeptide and the additional salt increases the yield. Finally, the AmyWDG195-related polypeptide is recovered and purified as desired.
3.2 Materials and Methods for Protein Purification
Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated AmyWDG195-related amylase-containing solution.
After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an amylase solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.
It is desirable to concentrate an AmyWDG195-related amylase-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order to collect the purified enzyme precipitate.
The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved using any of the techniques discussed herein or otherwise known in the art. Exemplary methods of concentration include but are not limited to rotary vacuum filtration and/or ultrafiltration.
Concentration may be performed using e.g., a precipitation agent, such as a metal halide precipitation agent. Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide and blends of two or more of these metal halides. The metal halide precipitation agent, sodium chloride, can also be used as a preservative.
The metal halide precipitation agent is used in an amount effective to precipitate the AmyWDG195-related polypeptide. The selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.
Generally, at least about 5% w/v (weight/volume) to about 25% w/v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w/v. Generally, no more than about 25% w/v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w/v. The optimal concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific AmyWDG195-related amylase and on its concentration in the concentrated enzyme solution.
Another alternative to effect precipitation of the enzyme is to use organic compounds. Exemplary organic compound precipitating agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of said organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.
Generally, the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitation agents can be, for example, linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds. Exemplary organic compounds are linear alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used. Additional organic compounds also include but are not limited to 4-hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both amylase preservative agents. For further descriptions, see, e.g., U.S. Pat. No. 5,281,526.
Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, AmyWDG195-related polypeptide concentration, precipitation agent concentration, and time of incubation.
The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing.
Generally, at least about 0.01% w/v of organic compound precipitation agent is added to the concentrated AmyWDG195-related polypeptide solution and usually at least about 0.02% w/v. Generally, no more than about 0.3% w/v of organic compound precipitation agent is added to the concentrated AmyWDG195-related polypeptide solution and usually no more than about 0.2% w/v.
The concentrated enzyme solution, containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be purified. Generally, the pH is adjusted at a level near the isoelectric point of the amylase. The pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pI) up to about 2.5 pH units above the isoelectric point.
The incubation time necessary to obtain a purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less about 10 hours and in most cases even about 6 hours.
Generally, the temperature during incubation is between about 4° C. and about 50° C. Usually, the method is carried out at a temperature between about 10° C. and about 45° C. (e.g., between about 20° C. and about 40° C.). The optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used.
The overall recovery of purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound. The agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.
After the incubation period, the purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further purification of the purified enzyme precipitate can be obtained by washing the precipitate with water. For example, the purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.
During fermentation, an AmyWDG195-related amylase accumulates in the culture broth. For the isolation and purification of the desired AmyWDG195-related amylase, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme purification. In one embodiment, the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column, and eluted to recover the enzyme-active fraction. For further purification, a conventional procedure such as ion exchange chromatography may be used.
Purified enzymes are useful for laundry and cleaning applications. For example, they can be used in laundry detergents and spot removers. They can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).
A more specific example of purification, is described in Sumitani, J. et al. (2000) Biochem. J. 350: 477-484 and is briefly summarized here. The enzyme obtained from 4 liters of a Streptomyces lividans TK24 culture supernatant was treated with (NH4)2SO4 at 80% saturation. The precipitate was recovered by centrifugation at 10,000×g (20 minutes and 4° C.) and re-dissolved in 20 mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl2. The solubilized precipitate was then dialyzed against the same buffer. The dialyzed sample was then applied to a Sephacryl S-200 column, which had previously been equilibrated with 20 mM Tris/HCl buffer, (pH 7.0), 5 mM CaCl2, and eluted at a linear flow rate of 7 cm/hr with the same buffer. Fractions from the column were collected and assessed for activity as judged by enzyme assay and SDS-PAGE. The protein was further purified as follows. A Toyopearl HW55 column (Tosoh Bioscience, Montgomeryville, Pa.; Cat. No. 19812) was equilibrated with 20 mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl2 and 1.5 M (NH4)2SO4. The enzyme was eluted with a linear gradient of 1.5 to 0 M (NH4)2SO4 in 20 mM Tris/HCL buffer, pH 7.0 containing 5 mM CaCl2. The active fractions were collected, and the enzyme precipitated with (NH4)2SO4 at 80% saturation. The precipitate was recovered, re-dissolved, and dialyzed as described above. The dialyzed sample was then applied to a Mono Q HR5/5 column (Amersham Pharmacia; Cat. No. 17-5167-01) previously equilibrated with 20 mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl2, at a flow rate of 60 mL/hour. The active fractions are collected and added to a 1.5 M (NH4)2SO4 solution. The active enzyme fractions were re-chromatographed on a Toyopearl HW55 column, as before, to yield a homogeneous enzyme as determined by SDS-PAGE. See J. Sumitani et al., “New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 α-amylase contributes to starch binding and raw starch degrading,” Biochem. J. 350: 477-484 (2000) for general discussion of the method and variations thereon.
For production scale recovery, an AmyWDG195-related amylase can be partially purified as generally described above by removing cells via flocculation with polymers. Alternatively, the enzyme can be purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be purified, and whole broth culture can be lysed and used without further treatment. The enzyme can then be processed, for example, into granules.
4. Cleaning Compositions
An aspect of the present compositions and methods is a cleaning composition that includes an AmyWDG195-related amylase as a component. An AmyWDG195-related amylase can be used as a component in detergent compositions for hand washing, laundry washing, dishwashing, and other hard-surface cleaning. Preferably, an AmyWDG195-related amylase is incorporated into detergents at or near a concentration conventionally used for amylase in detergents. For example, an AmyWDG195-related amylase may be added in amount corresponding to 0.00001-1 mg (calculated as pure enzyme protein) of α-amylase per liter of wash/diswash liquor. Exemplary formulations are provided herein, as exemplified by the following:
4.1 Laundry Detergent Composition
An AmyWDG195-related amylase may typically be a component of a detergent composition, as the only enzyme or with other enzymes including other amylolytic enzymes. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in, for example, GB Patent No. 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method disclosed in for example EP 238,216. Polyols have long been recognized as stabilizers of proteins, as well as improving protein solubility. See, e.g., Kaushik, J. K. et al. (2003) J. Biol. Chem. 278: 26458-65 and references cited therein; and Monica Conti, M. et al. (1997) J. Chromatography A 757: 237-245.
The detergent composition may be in any useful form, e.g., as powders, granules, pastes, or liquid. A liquid detergent may be aqueous, typically containing up to about 70% of water and 0% to about 30% of organic solvent. It may also be in the form of a compact gel type containing only about 30% water.
The detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic. The detergent will usually contain 0% to about 50% of anionic surfactant, such as linear alkylbenzenesulfonate (LAS); α-olefinsulfonate (AOS); alkyl sulfate (fatty alcohol sulfate) (AS); alcohol ethoxysulfate (AEOS or AES); secondary alkanesulfonates (SAS); α-sulfo fatty acid methyl esters; alkyl- or alkenylsuccinic acid; or soap. The composition may also contain 0% to about 40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (as described for example in WO 92/06154).
The detergent composition may additionally comprise one or more other enzymes, such as lipase, another amylolytic enzyme, cutinase, protease, cellulase, peroxidase, and/or laccase in any combination.
The detergent may contain about 1% to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst). The detergent may also be unbuilt, i.e. essentially free of detergent builder. The enzymes can be used in any composition compatible with the stability of the enzyme. Enzymes generally can be protected against deleterious components by known forms of encapsulation, for example, by granulation or sequestration in hydro gels. Enzymes, and specifically α-amylases, such as AmyWDG195 molecules, either with or without starch binding domains, can be used in a variety of compositions including laundry and dishwashing applications, surface cleaners, as well as in compositions for ethanol production from starch or biomass.
The detergent may comprise one or more polymers. Examples include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
The detergent may contain a bleaching system, which may comprise a H2O2 source such as perborate or percarbonate, which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, the bleaching system may comprise peroxyacids (e.g., the amide, imide, or sulfone type peroxyacids). The bleaching system can also be an enzymatic bleaching system, for example, perhydrolase, such as that described in International PCT Application WO 2005/056783.
The enzymes of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol; a sugar or sugar alcohol; lactic acid; boric acid or a boric acid derivative such as, e.g., an aromatic borate ester; and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.
The detergent may also contain other conventional detergent ingredients such as e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibiters, optical brighteners, or perfumes.
The pH (measured in aqueous solution at use concentration) is usually neutral or alkaline, e.g., pH about 7.0 to about 11.0.
Particular forms of detergent compositions comprising the AmyWDG195-related polypeptides can be formulated to include:
1) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 7% to about 12%; alcohol ethoxysulfate (e.g., C12-18 alcohol, 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., C16-18) about 1% to about 4%; alcohol ethoxylate (e.g., C14-15 alcohol, 7 EO) about 5% to about 9%; sodium carbonate (e.g., Na2CO3) about 14% to about 20%; soluble silicate (e.g., Na2O, 2SiO2) about 2 to about 6%; zeolite (e.g., NaAlSiO4) about 15% to about 22%; sodium sulfate (e.g., Na2SO4) 0% to about 6%; sodium citrate/citric acid (e.g., C6H5Na3O7/C6H8O7) about 0% to about 15%; sodium perborate (e.g., NaBO3H2O) about 11% to about 18%; TAED about 2% to about 6%; carboxymethylcellulose (CMC) and 0% to about 2%; polymers (e.g., maleic/acrylic acid, copolymer, PVP, PEG) 0-3%; enzymes (calculated as pure enzyme) 0.0001-0.1% protein; and minor ingredients (e.g., suds suppressors, perfumes, optical brightener, photobleach) 0-5%.
2) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 6% to about 11%; alcohol ethoxysulfate (e.g., C12-18 alcohol, 1-2 EO) or alkyl sulfate (e.g., C16-18) about 1% to about 3%; alcohol ethoxylate (e.g., C14-15 alcohol, 7 EO) about 5% to about 9%; sodium carbonate (e.g., Na2CO3) about 15% to about 21%; soluble silicate (e.g., Na2O, 2SiO2) about 1% to about 4%; zeolite (e.g., NaAlSiO4) about 24% to about 34%; sodium sulfate (e.g., Na2SO4) about 4% to about 10%; sodium citrate/citric acid (e.g., C6H5Na3O7/C6H8O7) 0% to about 15%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-6%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., suds suppressors, perfume) 0-5%.
3) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 5% to about 9%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO) about 7% to about 14%; Soap as fatty acid (e.g., C16-22 fatty acid) about 1 to about 3%; sodium carbonate (as Na2CO3) about 10% to about 17%; soluble silicate (e.g., Na2O, 2SiO2) about 3% to about 9%; zeolite (as NaAlSiO4) about 23% to about 33%; sodium sulfate (e.g., Na2SO4) 0% to about 4%; sodium perborate (e.g., NaBO3H2O) about 8% to about 16%; TAED about 2% to about 8%; phosphonate (e.g., EDTMPA) 0% to about 1%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 0-3%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., suds suppressors, perfume, optical brightener) 0-5%.
4) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 8% to about 12%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO) about 10% to about 25%; sodium carbonate (as Na2CO3) about 14% to about 22%; soluble silicate (e.g., Na2O, 2SiO2) about 1% to about 5%; zeolite (e.g., NaAlSiO4) about 25% to about 35%; sodium sulfate (e.g., Na2SO4) 0% to about 10%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-3%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., suds suppressors, perfume) 0-5%.
5) An aqueous liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 15% to about 21%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO or C12-15 alcohol, 5 EO) about 12% to about 18%; soap as fatty acid (e.g., oleic acid) about 3% to about 13%; alkenylsuccinic acid (C12-14) 0% to about 13%; aminoethanol about 8% to about 18%; citric acid about 2% to about 8%; phosphonate 0% to about 3%; polymers (e.g., PVP, PEG) 0% to about 3%; borate (e.g., B4O7) 0% to about 2%; ethanol 0% to about 3%; propylene glycol about 8% to about 14%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., dispersants, suds suppressors, perfume, optical brightener) 0-5%.
6) An aqueous structured liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 15% to about 21%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO, or C12-15 alcohol, 5 EO) 3-9%; soap as fatty acid (e.g., oleic acid) about 3% to about 10%; zeolite (as NaAlSiO4) about 14% to about 22%; potassium citrate about 9% to about 18%; borate (e.g., B4O7) 0% to about 2%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., PEG, PVP) 0% to about 3%; anchoring polymers such as, e.g., lauryl methacrylate/acrylic acid copolymer; molar ratio 25:1, MW 3800) 0% to about 3%; glycerol 0% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., dispersants, suds suppressors, perfume, optical brighteners) 0-5%.
7) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising fatty alcohol sulfate about 5% to about 10%; ethoxylated fatty acid monoethanolamide about 3% to about 9%; soap as fatty acid 0-3%; sodium carbonate (e.g., Na2CO3) about 5% to about 10%; Soluble silicate (e.g., Na2O, 2SiO2) about 1% to about 4%; zeolite (e.g., NaAlSiO4) about 20% to about 40%; Sodium sulfate (e.g., Na2SO4) about 2% to about 8%; sodium perborate (e.g., NaBO3H2O) about 12% to about 18%; TAED about 2% to about 7%; polymers (e.g., maleic/acrylic acid copolymer, PEG) about 1% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., optical brightener, suds suppressors, perfume) 0-5%.
8) A detergent composition formulated as a granulate comprising linear alkylbenzenesulfonate (calculated as acid) about 8% to about 14%; ethoxylated fatty acid monoethanolamide about 5% to about 11%; soap as fatty acid 0% to about 3%; sodium carbonate (e.g., Na2CO3) about 4% to about 10%; soluble silicate (Na2O, 2SiO2) about 1% to about 4%; zeolite (e.g., NaAlSiO4) about 30% to about 50%; sodium sulfate (e.g., Na2SO4) about 3% to about 11%; sodium citrate (e.g., C6H5Na3O7) about 5% to about 12%; polymers (e.g., PVP, maleic/acrylic acid copolymer, PEG) about 1% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., suds suppressors, perfume) 0-5%.
9) A detergent composition formulated as a granulate comprising linear alkylbenzenesulfonate (calculated as acid) about 6% to about 12%; nonionic surfactant about 1% to about 4%; soap as fatty acid about 2% to about 6%; sodium carbonate (e.g., Na2CO3) about 14% to about 22%; zeolite (e.g., NaAlSiO4) about 18% to about 32%; sodium sulfate (e.g., Na2SO4) about 5% to about 20%; sodium citrate (e.g., C6H5Na3O7) about 3% to about 8%; sodium perborate (e.g., NaBO3H2O) about 4% to about 9%; bleach activator (e.g., NOBS or TAED) about 1% to about 5%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., polycarboxylate or PEG) about 1% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., optical brightener, perfume) 0-5%.
10) An aqueous liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 15% to about 23%; alcohol ethoxysulfate (e.g., C12-15 alcohol, 2-3 EO) about 8% to about 15%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO, or C12-15 alcohol, 5 EO) about 3% to about 9%; soap as fatty acid (e.g., lauric acid) 0% to about 3%; aminoethanol about 1% to about 5%; sodium citrate about 5% to about 10%; hydrotrope (e.g., sodium toluensulfonate) about 2% to about 6%; borate (e.g., B4O7) 0% to about 2%; carboxymethylcellulose 0% to about 1%; ethanol about 1% to about 3%; propylene glycol about 2% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., polymers, dispersants, perfume, optical brighteners) 0-5%.
11) An aqueous liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 20% to about 32%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO, or C12-15 alcohol, 5 EO) 6-12%; aminoethanol about 2% to about 6%; citric acid about 8% to about 14%; borate (e.g., B4O7) about 1% to about 3%; polymer (e.g., maleic/acrylic acid copolymer, anchoring polymer such as, e.g., lauryl methacrylate/acrylic acid copolymer) 0% to about 3%; glycerol about 3% to about 8%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., hydrotropes, dispersants, perfume, optical brighteners) 0-5%.
12) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising anionic surfactant (linear alkylbenzenesulfonate, alkyl sulfate, α-olefinsulfonate, α-sulfo fatty acid methyl esters, alkanesulfonates, soap) about 25% to about 40%; nonionic surfactant (e.g., alcohol ethoxylate) about 1% to about 10%; sodium carbonate (e.g., Na2CO3) about 8% to about 25%; soluble silicates (e.g., Na2O, 2SiO2) about 5% to about 15%; sodium sulfate (e.g., Na2SO4) 0% to about 5%; zeolite (NaAlSiO4) about 15% to about 28%; sodium perborate (e.g., NaBO3.4H2O) 0% to about 20%; bleach activator (TAED or NOBS) about 0% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients (e.g., perfume, optical brighteners) 0-3%.
13) Detergent compositions as described in compositions 1)-12) supra, wherein all or part of the linear alkylbenzenesulfonate is replaced by (C12-C18) alkyl sulfate.
14) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising (C12-C18) alkyl sulfate about 9% to about 15%; alcohol ethoxylate about 3% to about 6%; polyhydroxy alkyl fatty acid amide about 1% to about 5%; zeolite (e.g., NaAlSiO4) about 10% to about 20%; layered disilicate (e.g., SK56 from Hoechst) about 10% to about 20%; sodium carbonate (e.g., Na2CO3) about 3% to about 12%; soluble silicate (e.g., Na2O, 2SiO2) 0% to about 6%; sodium citrate about 4% to about 8%; sodium percarbonate about 13% to about 22%; TAED about 3% to about 8%; polymers (e.g., polycarboxylates and PVP) 0% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., optical brightener, photobleach, perfume, suds suppressors) 0-5%.
15) A detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising (C12-C18) alkyl sulfate about 4% to about 8%; alcohol ethoxylate about 11% to about 15%; soap about 1% to about 4%; zeolite MAP or zeolite A about 35% to about 45%; sodium carbonate (as Na2CO3) about 2% to about 8%; soluble silicate (e.g., Na2O, 2SiO2) 0% to about 4%; sodium percarbonate about 13% to about 22%; TAED 1-8%; carboxymethylcellulose (CMC) 0% to about 3%; polymers (e.g., polycarboxylates and PVP) 0% to about 3%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., optical brightener, phosphonate, perfume) 0-3%.
16) Detergent formulations as described in 1)-15) supra, which contain a stabilized or encapsulated peracid, either as an additional component or as a substitute for already specified bleach systems.
17) Detergent compositions as described supra in 1), 3), 7), 9), and 12), wherein perborate is replaced by percarbonate.
18) Detergent compositions as described supra in 1), 3), 7), 9), 12), 14), and 15), which additionally contain a manganese catalyst. The manganese catalyst for example is one of the compounds described in Rage et al. (1994) Nature 369:637-639.
19) Detergent composition formulated as a non-aqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g., phosphate), an enzyme(s), and alkali. The detergent may also comprise anionic surfactant and/or a bleach system.
An AmyWDG195-related amylase may be incorporated in concentrations conventionally employed in detergents. It is at present contemplated that, in the detergent composition, the enzyme may be added in an amount corresponding to 0.00001-1.0 mg (calculated as pure enzyme protein) of AmyWDG195-related amylase per liter of wash liquor.
In another embodiment, other enzymes, such as 2,643-D-fructan hydrolase, can be incorporated in detergent compositions comprising an AmyWDG195-related amylase and used for removal/cleaning of biofilm present on household and/or industrial textile/laundry.
The detergent composition may for example be formulated as a hand (manual) or machine (automatic) laundry detergent composition, including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for manual or automatic dishwashing operations.
In a specific aspect, the detergent composition can comprise 2,6-β-D-fructan hydrolase in addition to an AmyWDG195-related amylase, and one or more other cleaning enzymes, such as a protease, a lipase, a cutinase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, another amylolytic enzyme, a xylanase, an oxidase, a laccase, and/or a peroxidase, and/or combinations thereof.
In general the properties of the chosen enzyme(s) should be compatible with the selected detergent, (e.g., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
Proteases:
Suitable proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are included, as well as naturally processed proteins. The protease may be a serine protease or a metalloprotease, such as an alkaline microbial protease, a trypsin-like protease, or a chymotrypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin), and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583). Examples of useful proteases also include but are not limited to the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946. Commercially available protease enzymes include but are not limited to: ALCALASE®, SAVINASE®, PRIMASETM, DURALASETM, ESPERASE®, and KANNASETM (Novo Nordisk A/S); MAXATASE®, MAXACALTM, MAXAPEMTTM, PROPERASE®, PURAFECT®, PURAFECT OXPTM, FN2TM, and FN3TM (Genencor International, Inc.).
Lipases:
Suitable lipases include those of bacterial or fungal origin. Chemically modified, proteolytically modified, or protein engineered mutants are included. Examples of useful lipases include but are not limited to lipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) (see e.g., EP 258068 and EP 305216), from H. insolens (see e.g., WO 96/13580); a Pseudomonas lipase (e.g., from P. alcaligenes or P. pseudoalcaligenes; see, e.g., EP 218 272), P. cepacia (see e.g., EP 331 376), P. stutzeri (see e.g., GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (see e.g., WO 95/06720 and WO 96/27002), P. wisconsinensis (see e.g., WO 96/12012); a Bacillus lipase (e.g., from B. subtilis; see e.g., Dartois et al. Biochemica et Biophysica Acta, 1131: 253-360 (1993)), Geobacillus (formerly Bacillus) stearothermophilus (see e.g., JP 64/744992), or B. pumilus (see e.g., WO 91/16422). Additional lipase variants contemplated for use in the formulations include those described for example in: WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, EP 407225, and EP 260105. Some commercially available lipase enzymes include LIPOLASE® and LIPOLASE ULTRATM (Novo Nordisk A/S).
Polyesterases:
Suitable polyesterases can be included in the composition, such as those described in, for example, WO 01/34899 and WO 01/14629.
Amylases:
The compositions can be combined with other amylases, such as non-production enhanced α-amylase. These can include commercially available amylases, such as but not limited to DURAMYL®, TERMAMYL®, FUNGAMYL® and BANTM (Novo Nordisk A/S); RAPIDASE® and PURASTAR® (from Genencor International, Inc.).
Cellulases:
Cellulases can be added to the compositions. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed for example in U.S. Pat. Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89/09259. Exemplary cellulases contemplated for use are those having color care benefit for the textile. Examples of such cellulases are cellulases described in for example EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, and WO 98/08940. Other examples are cellulase variants, such as those described in WO 94/07998; WO 98/12307; WO 95/24471; PCT/DK98/00299; EP 531315; U.S. Pat. Nos. 5,457,046; 5,686,593; and 5,763,254. Commercially available cellulases include CELLUZYME® and CAREZYME® (Novo Nordisk A/S); CLAZINASE® and PURADAX HA® (Genencor International, Inc.); and KAC-500(B)TM (Kao Corporation).
Peroxidases/Oxidases:
Suitable peroxidases/oxidases contemplated for use in the compositions include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include for example GUARDZYMETM (Novo Nordisk A/S).
The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive, i.e. a separate additive or a combined additive, can be formulated e.g., as a granulate, a liquid, a slurry, etc. Exemplary detergent additive formulations include but are not limited to granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids or slurries.
Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (e.g., polyethyleneglycol, PEG) with mean molar weights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in, for example, GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216.
The detergent composition may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste, or a liquid. A liquid detergent may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent. Compact detergent gels containing about 30% or less water are also contemplated. The detergent composition can optionally comprise one or more surfactants, which may be non-ionic, including semi-polar and/or anionic and/or cationic and/or zwitterionic. The surfactants can be present in a wide range, from about 0.1% to about 60% by weight.
When included therein the detergent will typically contain from about 1% to about 40% of an anionic surfactant, such as linear alkylbenzenesulfonate, α-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, α-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, or soap.
When included therein, the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl-N-alkyl derivatives of glucosamine (“glucamides”).
The detergent may contain 0% to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).
The detergent may comprise one or more polymers. Exemplary polymers include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates e.g., polyacrylates, maleic/acrylic acid copolymers), and lauryl methacrylate/acrylic acid copolymers.
The enzyme(s) of the detergent composition may be stabilized using conventional stabilizing agents, e.g., as polyol (e.g., propylene glycol or glycerol), a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative (e.g., an aromatic borate ester), or a phenyl boronic acid derivative (e.g., 4-formylphenyl boronic acid). The composition may be formulated as described in WO 92/19709 and WO 92/19708.
It is at present contemplated that in the detergent compositions, the AmyWDG195-related polypeptides may be added in an amount corresponding to about 0.01 to about 100 mg of enzyme protein per liter of wash liquor (e.g., about 0.05 to about 5.0 mg of enzyme protein per liter of wash liquor or 0.1 to about 1.0 mg of enzyme protein per liter of wash liquor).
4.2 Cleaning Compositions
In the detergent applications, an AmyWDG195-related amylase is usually used in a liquid composition containing propylene glycol. The enzyme is solubilized in for example in propylene glycol by mixing in a 25% volume/volume propylene glycol solution containing 10% calcium chloride.
An AmyWDG195-related amylase thereof discussed herein can be formulated in detergent compositions for use in cleaning dishes or other cleaning compositions. These can be powders, gels, or liquids. The compositions can comprise the enzyme alone, or with other amylolytic enzymes and/or with other cleaning enzymes or bleach activating enzymes, and other components common to cleaning compositions.
Thus, a dishwashing detergent composition can comprise a surfactant. The surfactant may be anionic, non-ionic, cationic, amphoteric or a mixture of these types. The detergent can contain 0% to about 90% by weight of a non-ionic surfactant, such as low- to non-foaming ethoxylated propoxylated straight-chain alcohols.
The detergent composition may contain detergent builder salts of inorganic and/or organic types. The detergent builders may be subdivided into phosphorus-containing and non-phosphorus-containing types. The detergent composition usually contains about 1% to about 90% of detergent builders. Examples of phosphorus-containing inorganic alkaline detergent builders, when present, include the water-soluble salts, especially alkali metal pyrophosphates, orthophosphates, and polyphosphates. An example of phosphorus-containing organic alkaline detergent builder, when present, includes the water-soluble salts of phosphonates. Examples of non-phosphorus-containing inorganic builders, when present, include water-soluble alkali metal carbonates, borates, and silicates, as well as the various types of water-insoluble crystalline or amorphous alumino silicates, of which zeolites are the best-known representatives.
Examples of suitable organic builders include the alkali metal; ammonium and substituted ammonium; citrates; succinates; malonates; fatty acid sulphonates; carboxymethoxy succinates; ammonium polyacetates; carboxylates; polycarboxylates; aminopolycarboxylates; polyacetyl carboxylates; and polyhydroxysulphonates.
Other suitable organic builders include the higher molecular weight polymers and co-polymers known to have builder properties, for example appropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acid copolymers, and their salts.
The cleaning composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type. Examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite, and hypobromite, as well as chlorinated trisodium phosphate. Examples of organic chlorine/bromine-type bleaches are heterocyclic N-bromo- and N-chloro-imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric, and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations such as potassium and sodium. Hydantoin compounds are also suitable.
The cleaning composition may contain oxygen bleaches, for example in the form of an inorganic persalt, optionally with a bleach precursor or as a peroxy acid compound. Typical examples of suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates, and perphosphates. Exemplary activator materials are TAED, and glycerol triacetate. Enzymatic bleach activation systems may also be present in the formulation, e.g., such as perborate or percarbonate, glycerol triacetate and perhydrolase (see, e.g., WO 2005/056783).
The cleaning composition may be stabilized using conventional stabilizing agents for the enzyme(s), e.g., a polyol such as, e.g., propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative (e.g., an aromatic borate ester).
The cleaning composition may also contain other conventional detergent ingredients, e.g., deflocculant material, filler material, foam depressors, anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bactericides, fluorescers, thickeners, and perfumes.
Although the present compositions and methods have been described with reference to the details below, it would be understood that various modifications can be made.
4.3 Methods of Assessing Detergent Compositions
Numerous α-amylase cleaning assays exist. Exemplary description of testing cleaning includes the following.
A “swatch” is a piece of material such as a fabric that has a stain applied thereto. The material can be, for example, fabrics made of cotton, polyester or mixtures of natural and synthetic fibers. The swatch can further be paper, such as filter paper or nitrocellulose, or a piece of a hard material such as ceramic, metal, or glass. For amylases, the stain is starch based, but can include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of these compounds.
A “smaller swatch” is a section of the swatch that has been cut with a single hole punch device, or has been cut with a custom manufactured 96-hole punch device, where the pattern of the multi-hole punch is matched to standard 96-well microtiter plates, or the section has been otherwise removed from the swatch. The swatch can be of textile, paper, metal, or other suitable material. The smaller swatch can have the stain affixed either before or after it is placed into the well of a 24-, 48- or 96-well microtiter plate. The “smaller swatch” can also be made by applying a stain to a small piece of material. For example, the smaller swatch can be a stained piece of fabric 5⁄8′′ or 0.25′′ in diameter. The custom manufactured punch is designed in such a manner that it delivers 96 swatches simultaneously to all wells of a 96-well plate. The device allows delivery of more than one swatch per well by simply loading the same 96-well plate multiple times. Multi-hole punch devices can be conceived of to deliver simultaneously swatches to any format plate, including but not limited to 24-well, 48-well, and 96-well plates. In another conceivable method, the soiled test platform can be a bead made of either metal, plastic, glass, ceramic, or other suitable material that is coated with the soil substrate. The one or more coated beads are then placed into wells of 96-, 48-, or 24-well plates or larger formats, containing suitable buffer and enzyme. In this case, supernatant can be examined for released soil either by direct absorbance measurement or after a secondary color development reaction. Analysis of the released soil might also be taken by mass spectral analysis. A further microscreening assay can be to deliver and secure a swatch, for example an indigo dyed denim, to a well of a multi-well plate, and add particles such as sand or larger particles such as for example garnet sieved to include particle 6 to 8, or 9 gauge, and agitate the plate so as to cause abrasion of the swatch by the added particles. This assay has found use in the assessment of cellulases in stone washing applications. The effectiveness of the enzyme can be judged by either color release (e.g., released indigo is dissolved in dimethylsulfoxide and absorbance at A600 nm is measured) to the reaction buffer or by reflectance measurements of the abraded swatch.
When, for example, untreated BMI (blood/milk/ink) swatches are washed in detergent without bleach, a large portion of the ink is released even without the help of a protease. Adding a protease leads to a small increase in ink release, which can be hard to quantify over the large background. The present compositions and methods provide a treatment protocol that allows one to control the degree of fixation of a stain. As a result, it is possible to produce swatches that, for example, release varying amounts of stain when washed in the absence of the enzyme being tested. The use of fixed swatches leads to a dramatic improvement of the signal-to-noise ratio in the wash assays. Furthermore, by varying the degree of fixation, one can generate stains that give optimum results under the various cleaning conditions.
Swatches having stains of known “strength” on various types of material are commercially available (EMPA, St. Gallen, Switzerland; wfk-Testgewebe GmbH, Krefeld Germany; or Center for Test Materials, Vlaardingen, The Netherlands) and/or can be made by the practitioner (Morris and Prato, Textile Research Journal 52(4): 280 286 (1982)). Other test swatches include but are not limited to blood/milk/ink (BMI) stain(s) on a cotton-containing fabric, a spinach stain on a cotton-containing fabric, or grass on a cotton-containing fabric, and chocolate/milk/soot on a cotton-containing fabric.
A BMI stain can be fixed to cotton with 0.0003% to 0.3% hydrogen peroxide. Other combinations include grass or spinach fixed with 0.001% to 1% glutaraldehyde, gelatin and Coomassie stain fixed with 0.001% to 1% glutaraldehyde, or chocolate, milk and soot fixed with 0.001% to 1% glutaraldehyde.
The swatch can also be agitated during incubation with the enzyme and/or detergent formulation. Wash performance data is dependent on the orientation of the swatches in the wells (horizontal versus vertical), particularly in the 96-well plate. This would indicate that mixing was insufficient during the incubation period. Although there are a number of ways to ensure sufficient agitation during incubation, a plate holder in which the microtiter plate is sandwiched between two plates of aluminum can be constructed. This can be as simple as placing, for example, an adhesive plate sealer over the wells then clamping the two aluminum plates to the 96-well plate with any type of appropriate, commercially available clamps. It can then be mounted in a commercial incubator shaker. Setting the shaker to about 400 rpm results in very efficient mixing, while leakage or cross-contamination is efficiently prevented by the holder.
Trinitrobenzenesulfonic acid (TNBS) can be used to quantify the concentration of amino groups in the wash liquor. This can serve as a measure of the amount of protein that was removed from the swatch (see e.g., Cayot and Tainturier, Anal. Biochem. 249: 184-200 (1997)). However, if a detergent or an enzyme sample leads to the formation of unusually small peptide fragments (for example, from the presence of peptidases in the sample), then one will obtain a larger TNBS signal, i.e., more “noise”.
Another means of measuring wash performance of blood/milk/ink or other stain is based on ink release. Proteolysis of protein on the swatches leads to the release of ink particles which can be quantified by measuring the absorbance of the wash liquor. The absorbance can be measured at any wavelength between 350 and 800 nm. The absorbance is measured at 410 nm or 620 nm. The wash liquor can also be examined to determine the wash performance on stains containing grass, spinach, gelatin or Coomassie stain. Exemplary wavelengths for these stains include 670 nm for spinach or grass and 620 nm for gelatin or Coomassie. For example, an aliquot of the wash liquor (typically 100-150 μL from a 96-well microplate, for example) is removed and placed in a cuvette or multiwell microplate. This is then placed in a spectrophotometer and the absorbance is read at an appropriate wavelength.
The system can also be used to determine an enhanced enzyme and/or detergent composition for dish washing, for example, using a blood/milk/ink stain on a suitable substrate such as cloth, plastic or ceramic.
In one aspect, the BMI stain is fixed to cotton by applying 0.3% hydrogen peroxide to the BMI/cotton swatch for 30 minutes at 25° C. or by applying 0.03% hydrogen peroxide to the BMI/cotton swatch for 30 minutes at 60° C. Smaller swatches of approximately 0.25′′ are cut from the BMI/cotton swatch and placed in the wells of a 96-well microtiter plate. Into each well, a known mixture of a detergent composition and an enzyme is placed. After placing an adhesive plate sealer onto the top of the microtiter plate, the microtiter plate is clamped to an aluminum plate and agitated on an orbital shaker at approximately 250 rpm for about 10 to 60 minutes. At the end of this time, the supernatants are transferred to wells in a new microtiter plate and the absorbance of the ink at 620 nm is measured. This can be similarly tested with spinach stains or grass stains fixed to cotton by applying 0.01% glutaraldehyde to the spinach/cotton swatch or grass/cotton swatch for 30 minutes at 25° C. The same can be done with chocolate, milk, and/or soot stains.
The present application is organized into a number of sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for different sections of the disclosure should not be construed as limiting.
In order to further illustrate the compositions and methods, and advantages thereof, the following specific examples are given with the understanding that they are illustrative rather than limiting.
EXAMPLES
Example 1
Identification of Halomonas variabilis WDG195 Alpha-Amylase from an Expression Library of Halomonas variablis WDG195
Strain WDG195 was obtained from Prof. Grant, University of Leicester, UK. This strain was isolated from soda soil on the shore of Mono Lake, Calif., USA. Partial sequencing of the 16S rRNA gene (429 bp) indicated that the strain belongs to the species Halomonas variabilis. The partial 16S rRNA gene sequence is shown in SEQ ID NO: 2.
Cells of Halomonas variabilis WDG195 were cultivated on an alkaline medium, for example using methods according to Duckworth et al., FEMS Microbiology Letters 19 (1996) 181-191. Genomic DNA (>4 μg) was prepared using MasterPure Gram Positive DNA purification Kit from Epicentre (http://www.epibio.com/item.asp?id=429), using the manufacturer's protocol (http://www.epibio.com/pdftechlit/209p1085.pdf). The resulting genomic DNA was used by Eurofins Medigenomix GmbH (Martinsried, Germany) for the construction of an expression library. Fragments of 3-5 kb were obtained by random shearing and the fragments were cloned into low copy number vector pRANGER-BTB3-Cat (Lucigen Corporation, Middleton, Wis. USA, DQ058731) (FIG. 1). The vector was first modified for TA shotgun cloning by Medigenomix.
Ligation mixtures were transformed into electrocompetent TOP10 E. coli cells (Invitrogen Carlsbad, Calif., USA) and plated on RBB plates. These agar plates contain 10 g yeast extract, 16 g tryptone, 5 g NaCl, 15 g agar, 25 mg chloramphenicol, and 5 g starch dyed with Remazol brilliant blue R (Fluka/Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) per litter. An E. coli clone that formed a halo on the RBB plate, indicating the presence of an amylase gene that originates from Halomonas variabilis WG195, was selected for further analysis. The insert was sequenced by Baseclear (Leiden, The Netherlands) using the primers pRANGER-FW (SEQ ID NO: 3) and pRANGER-RV (SEQ ID NO: 4), which hybridize to the pRANGER vector. Based on this initial sequence analysis, the complete sequence of the amyWDG195 gene (SEQ ID NO: 9) was determined using four internal primers (SEQ ID NO: 5, 6, 7 and 8). The amyWDG195 gene encodes a precursor protein of 502 amino acids (SEQ ID NO: 10). The precursor protein consist of a signal peptide of 22 amino acids (SEQ ID NO: 11) and a mature portion of 480 amino acids (AmyWDG195, SEQ ID NO: 12). The results of a BLAST search performed through NCBI suggested that AmyWDG195 belongs to the alpha-amylase superfamily (FIG. 2), with its nearest neighbor being AmyK38 from Bacillus sp. KSM-K38, with which it shares 79% sequence identity.
Example 2
Protein Expression of Halomonas variabilis WDG195 Alpha-Amylase
To express AmyWDG195 amylase in Bacillus subtilis, an expression construct/plasmid was made based on the pHPLT vector, which contains the thermostable amylase LAT promoter (pLAT) and the LAT signal peptide (pre LAT) of Bacillus licheniformis, followed by PstI and HpaI restriction sites to facilitate cloning (FIG. 3; see, e.g., U.S. Pat. Nos. 5,871,550 and 6,562,612; U.S. Pat. Pub. No. 20060014265; and Solingen et al. (2001) Extremophiles 5:333-341).
Halomonas strainWDG195 genomic DNA was prepared from 1 ml cell pellet of an overnight culture (Tryptic Soy Broth at 37° C.) using the MasterPure Gram Positive DNA purification Kit from Epicentre (http://www.epibio.com/item.asp?id=429) according to the manufacturer's protocol (http://www.epibio.com/pdftechlit/209p1085.pdf). The amyWDG195 amylase gene was amplified from the genomic DNA sample by PCR performed on a MJ Research PTC-200 thermocycler with Phusion High Fidelity DNA polymerase (Finnzymes OY, Espoo, Finland) according to the instructions of the manufacturer (annealing temperature of 55° C.). The two primers used were: PstI-Amy195-1-Fw (SEQ ID NO: 13) and HpaI-Amy195-Rv (SEQ ID NO: 14).
The resulting PCR fragment was digested with restriction enzymes PstI and HpaI, and incubated with pHPLT DNA (50 ng/μl, also digested with PstI and HpaI) in the presence of T4 DNA ligase according to the instructions of the supplier (Roche Applied Science, Indianapolis, Ind., USA). The ligation mixture was transformed into B. subtilis strain BG6006, which is deleted for nine proteases (i.e., degUHy32,oppA,DspoII3501, amyE::xy1RPxy1AcomK-ermC, DaprE, DnprE, Depr, DispA, Dbpr, Dvpr, DwprA, Dmpr-ybfJ, DnprB) [see, e.g., U.S. Pat. Pub Nos. 20050202535 and 20020182734 (WO 02/14490)]. The transformation mixture was plated on DifcoTM Heart Infusion Agar plates containing 10 mg/L neomycin and 0.5% starch dyed with Remazol brilliant blue R. A starch-halo-positive colony was selected and the presence of the AmyWDG195 amylase gene in pHPLT was confirmed using colony PCR and sequencing. For colony PCR, the transformed bacteria was resuspended in 100 μL of sterile water of which 1 μL was used in a PCR reaction containing Invitrogen Platinum Taq DNA Polymerase and two primers: pHPLT-F1 (SEQ ID NO: 15) and pHPLT-R1 (SEQ ID NO: 16).
PCR was performed at 95° C. for 2 minutes followed by 25 cycles at 95° C. for 0.5 minute, 55° C. for 1 minute, and 68° C. for 2 minutes. The reaction was stopped after a final incubation at 68° C. for 5 minutes. The PCR product was sequenced at BaseClear (Leiden, The Netherlands) using primers pHPLT-F1 (SEQ ID NO: 15), pHPLT-R1 (SEQ ID NO: 16) and Amy195.int.-Rv (SEQ ID NO: 17).
Sequence analysis confirmed the presence of the AmyWDG195 amylase coding sequence in the pHPLT-AmyWDG195 vector.
Growth of B. subtilis transformants harbouring the pHPLT-AmyWDG195 vector was performed in shake flasks containing 25 ml MBD medium (a MOPS based defined medium: Neidhardt et al. (1974) J. Bacteriol., 119: 736-747) and 10 mg/L neomycin. This growth resulted in the production of secreted AmyWDG195 amylase with starch hydrolyzing activity. Adding additional NaCl to the growth medium increases the production of secreted and active AmyWDG195 protein, as shown in the following Table.















Relative Amylase



Production Medium
Expression









MBD
 1x



+1% NaCl
 9x



+2% NaCl
16x



+3% NaCl
35x










Example 3
Identification, Cloning, and Expression of AmySWT282
An amylase (AmySWT282) similar to AmyWDG195 (−78% amino acid identity) was identified from the alkaliphilic Bacillus strain SWT282 (isolated on alkaline tomato puree agar from a sample provided by Dr. Dimitri Sorokin (Inst. of Microbiology, Moscow, Russia), which was collected from the Kalunda Steppe (north) around Karasuk in the Novosibirsk region of Russia). Initially, a small fragment of the AmySWT282 gene was amplified from genomic DNA of this strain using degenerate primers DVVMNH-Forward (5′-GAYGTNGTNATGAAYCAY-3′; SEQ ID NO: 25; encoding 128 possible amino acid sequences) and VTFVDNHD-Reverse (5′-TCRTGRTTITCIACRAANGTNAC-3′; SEQ ID NO: 26; encoding 256 possible amino acid sequences). 2 μL of genomic DNA template and 1 μM of each primer (final concentration) were combined with water to a final volume of 25 μL with a PCR bead (GE Healthcare). The cycling conditions were as follows: 95° C., 4′; 95° C. 1′; 50° C., 1′; 72° C., 1′30′′; 72° C., 5′; 4° C. A short DNA fragment of roughly 700 bp was amplified (as visualized on a 1.2% agarase gel) and sequenced. The DNA sequence of this fragment is shown in FIG. 9 as SEQ ID NO: 18. A BlastX search proved that this fragment indeed encodes an alpha-amylase fragment.
To identify as much of the AmySWT282 amylase gene as possible, degenerate primers close to the N and C terminus of the predicted AmySWT282 amylase gene fragment were designed, in particular, a degenerate forward primer corresponding to the to N-terminal MMQ F/Y FEW motif (5′-ATGATGCARTWYTTYGARTGG-3′; SEQ ID NO: 27; encoding 32 possible amino acid sequences) and reverse primers corresponding to the C-terminal VTIN G/A DGWG E/N F motif (5′-AAITYICCCCAICCRTCNSCRTTNGTNAC-3′; SEQ ID NO: 28) or NGGSVSVYVN motif (5′-TTIACRAAIACISWIACISWICCNCCRTT-3′; SEQ ID NO: 29) (FIG. 9). Degenerate PCR reactions were set up using the MMQFEW forward primer separately in combination with each of the reverse primers listed above. Two μL of genomic DNA template and 1 μM of each primer (final concentration) were combined with water to a final volume of 25 μl with a PCR bead (GE Healthcare). The cycling conditions were as follows: 95° C., 4′, 95° C., 1′; 50° C., 1′; 72° C., 1′30′′; 72° C., 5′; 4° C. A 2.5 μl sample of the PCR reaction was analyzed on a 1.2% agarose gel. The forward MMQFEW and reverse VTINGDGWGEF primers produced a 1.4 kb band on the gel.
The band was gel purified using Qiagen gel purification kit and the DNA was eluted in 35 μl of buffer EB. 4 μl of the purified fragment was cloned into the PCR2.1 TOPO® vector using a 30 min TOPO® cloning reaction (Invitrogen Corp., Carlsbad, Calif.) at room temp. 2.5 μl of the reaction was transformed into 50 μl of Top 10 chemically competent cells. After ice and heat shock, 250 μl of SOC media was added to the cells and sample shaken at 37° C. for 1 hour. After one hour, 200 μl of the cell suspension was plated on IPTG-X-gal plates containing 100 ppm carbenicillin and the plates were incubated at 37° C. overnight. 4 white colonies were picked from the X-gal plates and used for colony PCR with the TOPO F (5′-CACTTTATGCTTCCGGCTCGTATG-3′; SEQ ID NO: 40) and TOPO R primers (5′-TCGCCATTCAGGCTGCGCAACTG-3; SEQ ID NO: 41). Three of the colonies produced the desired 1.7 kb band, which was sequenced with the TOPO seq F and R primers. This process yielded extended sequence information about the gene encoding the AmySWT282 gene.
To obtain more sequence information relating to the full-length amySWT282 gene, the following four primers were designed based on the extended sequence information. These primers were used in two different PCR reactions and designed for use with the Takara LA in vitro Cloning Kit (Takara Bio USA, Madison, Wis.).









N-Term SWT282 primer 2



(SEQ ID NO: 30)



5′-CTAAATCATACAAGTCATAGGCACCATAGCCGAC-3′



 



N-Term SWT282 primer 1



(SEQ ID NO: 31)



5′-GCAGCTCCTGCTTTGTGCCGTACTTCGTG-3′



 



C-term SWT282 primer 2



(SEQ ID NO: 32)



5′-GACCACTGGGATATCGTTGGTTGGACGAG-3′



 



C-term SWT282 primer 1



(SEQ ID NO: 33)



5′-CACGCACGGATTATTCTTACGGCACACAG-3′






Following the manufacturer's protocol AmySWT282 genomic DNA was digested with XbaI and ligated to the XbaI cassette. The first PCR reaction for the N terminal portion of the gene were carried out with primers 282 N3 (5′-TACGCCGTTAAAATGGTACCAGTGCCAG-3′; SEQ ID NO: 36)+kit primer C1, while the C terminal PCR reaction was performed using primers 282 C1 (5′-CACGCACGGATTATTCTTACGGCACACAG-3′; SEQ ID NO: 37)+kit primer C1. PCR products were confirmed on a gel, and then diluted 10−1, 10−2, and 10−4 before use as template for nested PCR reactions on the respective ends. The N-terminal nested PCR reaction used primer pair 282 N5 (5′ ATTCACCTGCACCGCATCCACCGCTTCGG 3′; SEQ ID NO: 38)+kit primer C2, while the C terminal nested PCR used primer pair 282 C2 (5′-GACCACTGGGATATCGTTGGTTGGACGAG-3′; SEQ ID NO: 39)+kit primer C2. The resulting PCR products were gel purified and ligated into the Topo TA vector, then transformed into 50 μl of Top 10 chemically competent cells. 4 colonies each were picked for PCR with primers Topo F and TOPO R (described, above) and the PCR products were sequenced. This resulted in new sequence information of the SWT282 amylase gene.
The last piece of sequence information of the amySWT282 gene was retrieved by directly sequencing genomic DNA of Bacillus strain SWT282 (Fidelity systems, Cessna Avenue, Gaithersburg, Md.), thereby completing the sequence of the amySWT282 gene. The full-length amySWT282 gene and AmySWT282 (precursor) protein sequences are shown as SEQ ID NOs: 19 and NO: 22, respectively. AmySWT282 has a signal peptide (SEQ ID NO: 21 and NO: 24), and is thus an extracellular protein. The mature AmySWT282 polypeptide has 479 amino acid residues (SEQ ID NO: 23) and is encoded by the polynucleotide of SEQ ID NO: 20. The mature AmySWT282 polypeptides showed moderate homology (<61% identity) to the α-amylases from Bacillus licheniformis, Geobacillus (formerly Bacillus) stearothermophilus, and Bacillus amyloliquefaciens. 
The amySWT282 gene was cloned into the pHPLT B. subtilis expression vector as described for amyWDG195 in Example 2. A B. subtilis transformant harboring pHPLT-AmySWT282 was grown in shake flasks containing MBD medium with 10 mg/L neomycin, 20 mM extra CaCl2, and with and without additional NaCl (3%). After 72 h growth, culture supernatants were analyzed by SDS-PAGE (FIG. 6). The addition of extra NaCl to the growth medium was necessary for production of AmySWT282, as observed for AmyWDG195. In contrast, the addition of CaCl2 showed no beneficial affect on the heterologous expression of AmySWT282.
Example 4
Assay for Cleaning
Culture supernatant from the pHPLT-AmyWDG195 vector transformed cells grown in MBD medium (Example 2) was analyzed for activity in a 96-well CS28-orange-dyed-rice-starch-soiled-fabric-swatch micro-applications cleaning assay. Using a fabric punch, 1⁄4-inch discs were cut from CS28 colored rice starch stained fabric swatches. This starch includes a bound indicator dye to facilitate tracking (Test Fabrics Cat. No. CS-28; Center for Test Materials, Vlaardingen, The Netherlands). Two of these discs were placed in each well of a flat-bottomed 96-well assay plates.
For the α-amylase cleaning assay, a preselected buffer was added to the wells and equilibrated to a preselected temperature. For initial testing the buffers were 25 mM HEPES (pH 8.0) or 25 mM CAPS (pH 10.0), additionally containing 2 mM CaCl2 and 0.0005% TWEEN 80. About 200 μL of buffer was added to different wells and allowed to equilibrate at 40° C. 10-20 μL of diluted culture supernatant solution (final amylase concentration of 0 ppm to 2 ppm) were added to selected wells and the plates incubated with shaking at 1150 rpm for 1 hour. Enzyme cleaning performance was based on the amount of enzyme-dependent color released into the wash liquor. Color release was quantified spectro-photometrically at 488 nm by the transfer of 100 μL final wash liquor to a fresh microtiter plate. The data were plotted with the aid of GRAFIT from Erithicus Software. The data points were fitted with the Langmuir isotherm-fitting algorithm, which takes the same form as the Michaelis-Menten fitting algorithm.
The results of this assay demonstrated that AmyWDG195 is highly efficient at removing starchy stains from textile swatches at 40° C. at pH 8.0, but slightly less efficient at pH 10.0 (FIGS. 4 and 5).
Example 5
Megazyme Assay for α-Amylase Activity
The α-amylase activity of AmyWDG195 culture supernatant from the transformed cells grown on MBD medium (Example 2) was determined using the Ceralpha method (Megazyme International, Ireland), which was modified for use in 96-well microtiter plate. 25 μl blocked p-nitrophenyl maltoheptaoside (=BPNPG7) substrate, 5.45 mg/mL (Amylase HR Reagent, Catalogue Number R-AMHR4Megazyme International) was added to each well and pre-equilibrated for 10 minutes at 25° C. Diluted culture supernatant (25 μl) was transferred into the wells and mixed with the substrate. Dilutions were made in 50 mM MOPS buffer pH 7.15, with 50 mM NaCl and 0.1 mM CaCl2).
The plates were incubated with shaking at 650 rpm for 30 minutes in an incubator (iEMS, Labsystems), and the reaction was terminated by the addition of 50 μl 200 mM Boric acid/NaOH, pH 10.2, solution into each well. The absorbance at 400 nm indicated the level of α-amylase in the diluted sample.
All references cited herein are herein incorporated by reference in their entirety for all purposes.

## Claims

1. An isolated polypeptide having α-amylase activity and having at least 90% amino acid sequence identity to SEQ ID NO: 12 or SEQ ID NO: 23 and including at least one of the following amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23, as determined by amino acid sequence alignment: E at position 3, E at position 20, M at position 25, N at position 26, L at position 40, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, L at position 267, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, L at position 307, D at position 340, V at position 350, S at position 360, L at position 381, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479.

2. The polypeptide of claim 1, including:
(a) at least one of the following amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: E at position 3, E at position 20, M at position 25, N at position 26, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, D at position 340, V at position 350, S at position 360, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479;
(b) at least one of the following amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: M at position 25, N at position 26, L at position 177, H at position 179, E at position 294, T at position 298, and M at position 449;
(c) at least one of the following combinations of amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: M at position 25 and N at position 26, optionally with E at position 20; Q at position 91 and G at position 94; L at position 177, H at position 179, and G at position 181; D at position 280 and V at position 281, optionally with D at position 278; R at position 297 and T at position 298, optionally with E at position 294; E at position 386 and D at position 390, optionally with L at position 381; and M at position 449 and H at position 453;
(d) at least 30 glutamate residues;
(e) at least one of the following amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: N at position 104, D at position 194, N at position 200, and H at position 235;
(f) at least one of the following amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: Y at position 302, G at position 300, N at position 427, D at position 404, and W at position 403;
(g) at least one of the following amino acid residues at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: N at position 289, V at position 324, D at position 325, and S at position 337; or
(h) the following amino acid residue at the indicated amino acid position relative to SEQ ID NO: 12 or SEQ ID NO: 23: L at position 197.

3. The polypeptide of claim 1, wherein the activity is independent of calcium.

4. A cultured cell material comprising a polypeptide of claim 1.

5. A cleaning composition comprising the polypeptide of claim 1.

6. The polypeptide of claim 1, wherein the polypeptide is a variant polypeptide that includes a man-made substitution, insertion, or deletion at one or more amino acid positions.

7. The polypeptide of claim 2, wherein the polypeptide is a variant polypeptide that includes a man-made substitution, insertion, or deletion at one or more amino acid positions.

8. The polypeptide of claim 3, wherein the polypeptide is a variant polypeptide that includes a man-made substitution, insertion, or deletion at one or more amino acid positions.

9. A cultured cell material comprising a polypeptide of claim 6.

10. A cleaning composition comprising the polypeptide of claim 6.