HK1166819A - Bacillus megaterium strain dsm90-related alpha-amylases, and methods of use, thereof - Google Patents

Description

Bacillus megaterium strain DSM90 related alpha-amylase and method of use thereof

Priority

Priority of united states provisional patent application serial No. 61/158,950, filed 3, 10, 2009, the application is hereby incorporated by reference in its entirety.

Technical Field

Compositions and methods relating to alpha-amylase obtained from Bacillus megaterium strain DSM90 and to structurally related amylases are disclosed.

Background

The starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w). Amylose consists of a linear chain of alpha-1, 4-linked glucose units having a Molecular Weight (MW) of from about 60,000 to about 800,000. Amylopectin is a branched polymer containing alpha-1, 6 branch points every 24-30 glucose units and can have a molecular weight of up to 1 hundred million.

Sugars from starch in the form of concentrated glucose syrups are currently produced by an enzymatic process that includes (1) liquefying (or viscosity-reducing) solid starch with alpha-amylase to dextrins having an average degree of polymerization of about 7-10, and (2) saccharifying the resulting liquefied starch (i.e., starch hydrolysate) with amyloglucosidase (also known as glucoamylase or GA). The resulting syrup has a high glucose content. Many glucose syrups produced commercially are subsequently enzymatically isomerized to a glucose/fructose mixture known as iso-syrup (isosyrup).

Alpha-amylases (EC 3.2.1.1) hydrolyze starch, glycogen and related polysaccharides by randomly cleaving internal alpha-1, 4-glucosidic bonds. Alpha-amylases, in particular those from the genus bacillus (bacillus), have been used for a number of different purposes, including starch liquefaction, fabric 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. There is a need for alpha-amylases having excellent cleaning properties.

Summary of The Invention

The compositions and methods of the present invention relate to alpha-amylase from bacillus megaterium strain DSM90 and related alpha-amylases, which represent a unique family of amylases for industrial applications. These amylases are collectively referred to as an AmyDSM 90-related polypeptide or an AmyDSM 90-related amylase.

In one aspect, an isolated polypeptide having at least 80% amino acid sequence identity to AmyDSM90(SEQ ID NO: 1), wherein the polypeptide has at least one amino acid sequence identity to SEQ ID NO: 1 has at least one of the following features:

a) an aspartic acid at position 21 of a compound of formula (I),

b) asparagine at position 97, and

c) isoleucine at position 128.

In some embodiments, the polypeptide has an aspartic acid at position 21 and an asparagine at position 97. In some embodiments, the polypeptide has an aspartic acid at position 21 and an isoleucine at position 128. In some embodiments, the polypeptide has an asparagine at position 97 and an isoleucine at position 128. In some embodiments, the polypeptide has an aspartic acid at position 21, an asparagine at position 97, and an isoleucine at position 128.

In some embodiments, the polypeptide is expressed as a secreted polypeptide by a heterologous cell.

In some embodiments, the polypeptide has alpha-amylase activity.

In some embodiments, the polypeptide hybridizes to SEQ ID NO: 1 has at least 90% identity. In some embodiments, the polypeptide hybridizes to SEQ ID NO: 1 has at least 95% identity. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

In a related aspect, isolated polypeptides are provided having the amino acid sequence of SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25 or SEQ ID NO: 26.

In yet another aspect, there is provided a composition comprising any one of the aforementioned polypeptides.

In some embodiments, the composition is a cleaning composition. In some embodiments, the composition is effective for removing starchy stains from laundry. In some embodiments, the composition is effective for removing starchy stains from dishware. In some embodiments, the composition is effective for removing starchy stains from fabrics.

In a related aspect, compositions are provided comprising a polypeptide having the sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

In some embodiments, the composition is a cleaning composition. In some embodiments, the composition is effective for removing starchy stains from laundry. In some embodiments, the composition is effective for removing starchy stains from dishware. In some embodiments, the composition is effective for removing starchy stains from fabrics.

In another aspect, a method for removing starchy stains from a surface is provided, the method comprising

Incubating the surface in the presence of an aqueous composition comprising an effective amount of an alpha-amylase having at least 80% amino acid sequence identity to AmyDSM90(SEQ ID NO: 1), wherein the polypeptide has an amino acid sequence as compared to the amino acid sequence of SEQ ID NO: 1 has at least one of the following features:

a) an aspartic acid at position 21 of a compound of formula (I),

b) asparagine at position 97, and

c) isoleucine at position 128;

allowing the alpha-amylase to hydrolyze a starch component present in the starchy stain to produce starch-derived smaller molecules that are solubilized in the aqueous composition,

thereby removing starchy stains from the surface.

In some embodiments, the alpha-amylase is AmyDSM90(SEQ ID NO: 1). In some embodiments, the alpha-amylase is a variant AmyDSM90(SEQ ID NO: 28) having a deletion of residues R179 and G180. In some embodiments, the alpha-amylase is a variant of AmyDSM90(SEQ ID NO: 29) having a substitution at M200. In some embodiments, the alpha-amylase is an AmyDSM90 variant (SEQ ID NO: 30) having deletions at residues R179 and G180 and a substitution at M200.

In another aspect, a method for removing starchy stains from a surface is provided, the method comprising

Incubating the surface in the presence of an aqueous composition comprising an effective amount of a peptide having SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

In some embodiments, the surface is a fabric surface. In some embodiments, the surface is on a dish. In some embodiments, the surface is a garment surface.

In another aspect, there is provided a method for expressing an alpha-amylase, the method comprising:

introducing into a host cell an expression vector comprising a polynucleotide encoding an alpha-amylase having at least 80% amino acid sequence identity to AmyDSM90(SEQ ID NO: 1), wherein the polynucleotide is fused in-frame to a signal sequence;

expressing the alpha-amylase as a secreted polypeptide into the host cell culture medium; and

recovering the secreted alpha-amylase from the host cell growth medium;

thus isolating the alpha-amylase as a secreted polypeptide.

In some embodiments, the signal sequence is a native signal sequence. In some embodiments, the signal sequence is from Bacillus (Bacillus) AmyE or AprE or Streptomyces (Streptomyces) CelA.

In some embodiments, the α -amylase has the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

In some embodiments, the coding sequence and the sequence encoding an alpha-amylase together have the same sequence as SEQ id no: 10 has at least 80% identity.

In some embodiments, the polypeptide has a sequence relative to SEQ ID NO: 1 has at least one of the following features:

a) an aspartic acid at position 21 of a compound of formula (I),

b) asparagine at position 97, and

c) isoleucine at position 128.

In another aspect, polynucleotides encoding the aforementioned polypeptides are provided, as well as expression vectors comprising such polynucleotides. In yet another aspect, host cells comprising these expression vectors are provided.

These and other aspects and embodiments of compositions and methods will be apparent from the description of the invention and the accompanying drawings.

Brief Description of Drawings

FIG. 1 alignment of protein sequences from Bacillus megaterium DSM90(AmyDSM90, SEQ ID NO: 1) and Bacillus megaterium AAK00598(SEQ ID NO: 3).

FIG. 2 alignment of orthologous amylase sequences from Bacillus megaterium DSM90(AmyDSM90, SEQ ID NO: 1), Bacillus anthracis (B.antrhrasis) AAT32659(SEQ ID NO: 4), Bacillus thuringiensis (B.thuringiensis) AAT60457(SEQ ID NO: 5), Bacillus cereus (B.cereus) AAP10417(SEQ ID NO: 6) and Bacillus megaterium AAK00598(SEQ ID NO: 3).

FIG. 3. schematic representation of the plasmid vector pHPLT containing the thermostable amylase LAT promoter (pLAT) and the Bacillus licheniformis LAT signal peptide (pre LAT) followed by PstI and HpaI restriction sites for cloning.

FIG. 4 schematic representation of the plasmid pME602.13 (also known as pHPLT-B. meg DSM90Amy) containing the amylase gene amplified from the strain DSM 90.

FIG. 5 genetic constructs for AmyDSM90 expression.

FIG. 6 nucleotide (SEQ ID NO: 9) and corresponding amino acid sequence (SEQ ID NO: 10) of AmyDSM90 expressed in Bacillus licheniformis as a fusion protein with the Bacillus licheniformis alpha-amylase (LAT) signal peptide.

FIG. 7 is a schematic representation of pICAtH-B.meg DSM90amy plasmid containing LAT-DSM90 amylase gene.

FIG. 8 is a graph showing the cleaning performance of AmyDSM90 in 25mM BTP pH 8 buffer at 20 ℃ for 1 hour using CS-28 rice stained fabric samples.

FIG. 9 is a graph showing the cleaning performance of AmyDSM90 in 25mM BTP pH 8 buffer at 40 ℃ for 1 hour using CS-28 rice stained fabric samples.

FIG. 10 is a graph showing the cleaning performance of AmyDSM90 in 25mM CAPS pH 10.3 buffer at 20 ℃ for 1 hour using CS-28 rice stained fabric samples.

FIG. 11 is a graph showing the cleaning performance of AmyDSM90 in 25mM CAPS pH 10.3 buffer at 40 ℃ for 1 hour using CS-28 rice stained fabric samples.

FIG. 12 is a graph showing the cleaning of CS-28 rice starch stained fabric samples with AmyDSM 90-related polypeptide in 25mM HEPES buffer, pH 8.

FIG. 13 is a graph showing the cleaning of CS-28 rice stained fabric samples with AmyDSM 90-related polypeptide in 25mM CAPS buffer, pH10.

FIG. 14 is a graph showing the laundry wash performance of Bacillus megaterium amylase in AATCC powder detergent, pH 10.5, using samples of fabric stained with CS-28 rice starch.

FIG. 15 is a graph showing the laundry wash performance of Bacillus megaterium amylase in AATCC liquid detergent, pH 7, using samples of fabric stained with CS-28 rice starch.

FIG. 16 shows a plot of AmyDSM90 cleaning performance as a function of dose in a Terg-o-meter experiment at 40 ℃ in AATCC powdered detergent, pH10, using CS-28 rice starch stained fabric samples.

FIG. 17 is a graph showing the wash performance of Amy DSM90 in commercial gel detergents compared to commercial amylase.

FIG. 18 is a graph showing the wash performance of Amy DSM90 in a standard detergent compared to a commercial amylase.

FIG. 19 is a graph showing wash performance of AmyDSM 90-related amylase in commercial gel detergents.

FIG. 20 is a graph showing the wash performance of AmyDSM 90-related amylase in standard bleach-containing detergents.

FIG. 21 is a graph showing the wash performance of AmyDSM 90-related amylase in standard bleach-free detergent.

FIG. 22 is a polypeptide sequence and a nucleotide sequence referred to in the specification.

Brief description of the sequences

SEQ ID NO: 1 is the amino acid sequence of the mature amylase from bacillus megaterium AmyDSM 90.

SEQ ID NO: 2 is the nucleotide sequence of clone pME602.

SEQ ID NO: 3 is the amino acid sequence of mature amylase of bacillus megaterium AAK 00598.

SEQ ID NO: 4 is the amino acid sequence of bacillus anthracis AAT32659 mature amylase.

SEQ ID NO: 5 is the amino acid sequence of mature amylase of Bacillus thuringiensis AAT 60457.

SEQ ID NO: 6 is the amino acid sequence of Bacillus cereus AAP10417 mature amylase.

SEQ ID NO: 7 is the amino acid sequence of the LAT signal peptide of bacillus licheniformis.

SEQ ID NO: 8 is a partial nucleotide sequence of Bacillus megaterium DSM9016S rRNA.

SEQ ID NO: 9 is a nucleotide sequence of a polynucleotide encoding AmyDSM90 expressed in bacillus licheniformis as a fusion protein with the bacillus licheniformis alpha-amylase (LAT) signal peptide.

SEQ ID NO: 10 is the amino acid sequence of the AmyDSM90 polypeptide expressed in bacillus licheniformis as a fusion protein with the bacillus licheniformis alpha-amylase (LAT) signal peptide.

SEQ ID NOs: 11 and 12 are the nucleotide sequences of the PCR primers used to amplify the amyDSM90 gene from Bacillus megaterium DSM 90.

SEQ ID NOs: 13 and 14 are the nucleotide sequences of the PCR primers used in colony PCR to confirm the presence of the desired plasmid DNA sequence.

SEQ ID NOs: 15 and 16 are the nucleotide sequences of the sequencing primers used to confirm the presence of the desired plasmid DNA sequence.

SEQ ID NOs: 17 and 18 are nucleotide sequences of PCR primers used to amplify amyDSM90 related sequences to make Bacillus licheniformis expression strains.

SEQ ID NOs: 19 and 20 are nucleotide sequences of primers used to introduce the M200L substitution using the QUIK-CHANGE method.

SEQ ID NOs: 21 and 22 are the nucleotide sequences of primers used to introduce Δ RG deletions using the QUIK-CHANGE method.

SEQ ID NO: 23 is the nucleotide sequence of the synthetic gene encoding amylase from Bacillus anthracis AAT 32659.

SEQ ID NO: 24 is a nucleotide sequence of a synthetic gene encoding an amylase from bacillus cereus AAP 10417.

SEQ ID NO: 25 is the nucleotide sequence of the synthetic gene encoding amylase from bacillus thuringiensis AAT 60457.

SEQ ID NO: 26 is a nucleotide sequence encoding a synthetic gene for an amylase from Bacillus megaterium AAK 00598.

SEQ ID NO: 27 is the amino acid sequence of bacillus megaterium AAK00598 immature amylase. The signal peptide sequence is shown in bold.

SEQ ID NO: 28 is the amino acid sequence of AmyDSM90 amylase with Δ RG deletion.

SEQ ID NO: 29 is the amino acid sequence of AmyDSM90 amylase with a substitution of M200L.

SEQ ID NO: 30 is the amino acid sequence of AmyDSM90 amylase with Δ RG deletion and M200L substitution.

Detailed Description

Compositions and methods relating to alpha-amylase isolated from bacillus megaterium strain DSM90 and to structurally related amylases are described. These amylases are collectively referred to as an AmyDSM 90-related amylase or an AmyDSM 90-related polypeptide. AmyDSM90 is a hitherto undescribed secreted amylase that has several unique structural features that distinguish it from related amylases. In addition, the discovery that the AmyDSM 90-related polypeptides are secreted polypeptides rather than cytoplasmic polypeptides enables large-scale expression and purification of a variety of AmyDSM 90-related polypeptides for industrial and commercial use. An exemplary use of these amylases is in the preparation of cleaning compositions, such as detergent compositions, for laundry, dishware, fabrics, and other surfaces. These and other aspects of the compositions and methods are described in more detail below.

1.Definitions and abbreviations

The following abbreviations and definitions apply in light of this detailed description. It should be noted 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 dose" includes reference to one or more doses 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. For clarity, the following abbreviations and/or terms are defined.

1.1Acronyms/acronyms

The following abbreviations/acronyms have the following meanings, unless otherwise indicated:

AE fatty alcohol ethoxylates

AEO fatty alcohol ethoxylates

AEOS fatty alcohol ethoxylated sulfates

AES fatty alcohol ethoxylated sulfate

AmyDSM90 alpha-amylase from Bacillus megaterium strain DSM90

AOS alpha-olefinsulfonic acid ester

AS alkyl sulfates

CBD-25 carbohydrate binding domain protein family 25

cDNA complementary DNA

CMC carboxymethyl cellulose

DNA deoxyribonucleic acid

DTMPA Diethylenetriamine Pentacetic acid

EC enzyme Committee

EDTA ethylene diamine tetraacetic acid

EMPAMaterialprüfungs-und Forschungs

Antaltalt (Swiss federal materials testing and research laboratory)

EO Oxirane (Polymer segment)

F & HC Fabric and household Care

GA glucoamylase

IPTG isopropyl beta-D-thiogalactoside

kDa kilodalton

LAS linear alkylbenzenesulfonate

LAT Bacillus licheniformis (B.licheniformis) amylase

MW molecular weight

MWU modified Wohlgemuth units; 1.6x10^-5mg/MWU ═ activity unit

Nobs nonanoyloxy benzene sulfonate

NTA nitriloacetic acid

OxAm Purastar HPAM 5000L(Genencor International，Inc.)

PEG polyethylene glycol

pI isoelectric point

PVA poly (vinyl alcohol)

PVP poly (vinylpyrrolidone)

RNA ribonucleic acid

SAS alkylsulfonic acid salts

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% wt

DEG C

H₂O water

dH₂O or DI deionized water

dIH₂O Milli-Q filtered deionized water

g or gm gram

Microgram of μ g

mg of

kg kilogram

μ L and μ L microliter

mL and mL

mm

Micron diameter of

M mol

mM millimole

Micromolar μm

U unit

sec second

min for

hr hour

DO dissolved oxygen

Genencor Danisco US Inc，Genencor Division，Palo Alto，CA

Ncm Newton cm

ETOH ethanol

eq. equivalent

N normal

DS or DS dry solids content

1.2Definition of

The term "amylase" or "amylolytic enzyme" refers to an enzyme that is capable of catalyzing, inter alia, the degradation of starch. Amylases are hydrolases that cleave α -D- (1 → 4) O-glycosidic bonds in starch. In general, alpha-amylases (EC 3.2.1.1; alpha-D- (1 → 4) -glucan glucohydrolases) are defined as endonucleases that cleave the alpha-D- (1 → 4) O-glycosidic bond within the starch molecule in a random fashion. In contrast, exo-acting amylolytic enzymes, such as β -amylases (EC 3.2.1.2; α -D- (1 → 4) -glucanmaltohydrolase) and some product-specific amylases such as maltogenic α -amylase (EC 3.2.1.133), cleave starch molecules from the non-reducing ends of the substrate. Beta-amylases, alpha-glucosidases (EC 3.2.1.20; alpha-D-glucosinolate glucohydrolases), glucoamylases (EC 3.2.1.3; alpha-D- (1 → 4) -glucan glucohydrolases) and product-specific amylases can produce maltooligosaccharides of specific length from starch.

As used herein, the term "starch" refers to any substance comprising complex polysaccharide carbohydrates of plants, comprising a polysaccharide having the formula (C)₆H₁₀O₅)_xThe amylose and amylopectin of (1), wherein X may be any number. The term includes radicalsPlant-derived materials such as cereals, pasture grasses, tubers and roots, and more specifically include materials obtained from wheat, barley, cereals, rye, rice, sorghum, bran, cassava (cassava), millet, potatoes, sweet potatoes and tapioca (tapioca).

With respect to polypeptides, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polypeptide that does not include artificial substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polynucleotide that does not include artificial nucleoside alterations. It should be noted, however, that a polynucleotide encoding a wild-type, parent or reference polypeptide is not limited to a naturally occurring polynucleotide, but includes any polynucleotide encoding a wild-type, parent or reference polypeptide.

With respect to a polypeptide, the term "variant" refers to a polypeptide that differs from a specified wild-type, parent or reference polypeptide in that the polypeptide includes artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the term "variant" refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parent or reference polynucleotide. The identity of the wild-type, parent or reference polypeptide or polynucleotide will be apparent from the 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 conditions different than those found in nature.

The terms "recovering", "isolating" and "separating" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified substance or component that is removed from at least one other substance or component with which it is naturally associated as it occurs in nature.

As used herein, the term "purified" means a 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.

In the case of enzymes, the terms "thermostable" and "thermostability" refer to the ability of an enzyme to retain activity after exposure to elevated temperatures. Thermostability of enzymes (e.g.Amylase) the half-life (t.sub.t) of which is given in minutes, hours or days_1/2) Measured, half of the enzyme activity is lost under defined conditions during the half-life. The half-life can be calculated by measuring the residual alpha-amylase activity after exposure to (i.e. exposure to) elevated temperature.

In the case of an enzyme, the "pH range" means the range of pH values at which the enzyme exhibits catalytic activity.

As used herein, the terms "pH stable" and "pH stability" in reference to an enzyme relate to the ability of the enzyme to retain activity over a wide pH range for a predetermined period of time (e.g., 15 minutes, 30 minutes, 1 hour).

As used herein, "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and is used interchangeably. In the case where such amino acid sequences exhibit activity, they may be referred to as "enzymes". The conventional single or three letter code for amino acid residues is used, with the amino acid sequence being expressed in the standard amino to carboxy terminal orientation (i.e., N → C).

The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid may be single-stranded or double-stranded, and may be a chemical modification. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the compositions and methods of the invention include nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are expressed in the 5 '-to-3' direction.

"homolog" shall mean an entity having a specified degree of identity to the subject amino acid sequence and the subject nucleotide sequence. Homologous sequences are understood to include amino acid sequences that are 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, etc.). In general, homologues will comprise the same active site residues as the subject amino acid sequence, unless otherwise specified.

As used herein, the term "hybridization" refers to the process of base pairing of one strand of a nucleic acid to a complementary strand, which occurs during the blot hybridization technique and the PCR technique. Stringent hybridization conditions are exemplified by the following: 50 ℃ and 0.2 XSSC (1 XSSC ═ 0.15M NaCl, 0.015M trisodium citrate, pH 7.0). Highly stringent hybridization conditions are exemplified by the following: 65 ℃ and 0.1 XSSC (1 XSSC ═ 0.15M NaCl, 0.015M trisodium citrate, pH 7.0) ].

As used herein, a "synthetic" molecule is synthesized chemically or enzymatically in vitro and is not produced by an organism.

As used herein, the terms "transformed," "stably transformed," and "transgenic" when used in reference to a cell mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome, which is maintained over multiple generations.

The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection", "transformation" or "transduction", as is known in the art.

A "host strain" or "host cell" is an organism into which has been introduced an expression vector, phage, virus, or other DNA construct comprising a polynucleotide encoding a polypeptide of interest (e.g., a variant amylase). Exemplary host strains are bacterial cells. The term "host cell" includes protoplasts produced from cells, such as those of a species in the genus Bacillus.

The term "heterologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell.

The term "endogenous," with respect to a polynucleotide or protein, refers to a polynucleotide or protein that is naturally present in the host cell.

As used herein, the term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes transcription and translation.

"selectable marker" or "selectable marker" refers to a gene that can be 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 (e.g., a nutritional advantage) on the host cell.

"vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

By "expression vector" is meant a DNA construct comprising a DNA sequence encoding a polypeptide of interest, wherein the coding sequence is operably linked to suitable control sequences capable of effecting the expression of the DNA in a suitable host. Such regulatory sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences which control termination of transcription and translation.

The term "operatively connected" means that the specified components are in a relationship (including, but not limited to, being side-by-side) that allows them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequences.

A "signal sequence" is an amino acid sequence linked to the amino-terminal portion of a protein that facilitates secretion of the protein extracellularly. The mature form of the extracellular protein lacks a signal sequence that is cleaved off during the secretion process.

As used herein, "biologically active" refers to a sequence having a specified biological activity (e.g., enzymatic activity).

"Water hardness" is a measure of the minerals (e.g., calcium and magnesium) present in water.

As used herein, "cultured cell material comprising an AmyDSM 90-related polypeptide" or similar language refers to a cell lysate or supernatant (including culture medium) that includes an AmyDSM 90-related polypeptide as a component. The cellular material is preferably derived from a heterologous host grown in culture for the production of an AmyDSM 90-related polypeptide.

2.AmyDSM 90-related polypeptides and nucleic acids

One aspect of the compositions and methods of the invention is an AmyDSM 90-related polypeptide. The polypeptide may correspond to AmyDSM90, an amylase having a specified degree of identity to AmyDSM90, a variant of AmyDSM90 that includes an artificial substitution, insertion, or deletion, or a chimera thereof. An exemplary AmyDSM90 polypeptide has the amino acid sequence of SEQ ID NO: 1. The additional polypeptide has 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 the AmyDSM90 polypeptide.

In some embodiments, the AmyDSM90 polypeptide (relative to SEQ ID NO: 1) has at least one of the following characteristics: (a) an aspartic acid at position 21, (b) an asparagine at position 97, or (c) an isoleucine at position 128. The specified amino acid residues at these positions distinguish the AmyDSM90 polypeptide from orthologous amylases from Bacillus anthracis AAT32659(SEQ ID NO: 4), Bacillus thuringiensis AAT60457(SEQ ID NO: 5), Bacillus cereus AAP10417(SEQ ID NO: 6) and Bacillus megaterium AAK00598(SEQ ID NO: 3) (see, e.g., FIG. 2). Notably, the presence of aspartate at position 21 and asparagine at position 97 in the AmyDSM90 polypeptide is in contrast to the reverse arrangement observed in the orthologous amylase (i.e., asparagine is present at position 21 and aspartate is present at position 97). Without being bound by theory, it is suggested that the presence of a positively charged amino acid residue at position 21 is generally important for enzymatic activity, and that AmyDSM90 polypeptides have overcome the need for such charge at position 21 by employing a positively charged amino acid residue at position 97. However, this theory does not exclude the possibility of: each of the above-identified features that distinguish the AmyDSM90 polypeptide from an orthologous amylase are independent and independent.

Thus, in some embodiments, the AmyDSM90 polypeptide has only one of the above-identified characteristics, i.e., aspartic acid at position 21, asparagine at position 97, or isoleucine at position 128. In other embodiments, the α -amylase has two of the above-identified characteristics, namely, aspartic acid at position 21 in combination with asparagine at position 97, aspartic acid at position 21 in combination with isoleucine at position 128, or asparagine at position 97 in combination with isoleucine at position 128. In a specific embodiment, the alpha-amylase has all three of the above-identified characteristics, i.e., aspartic acid at position 21, asparagine at position 97, and isoleucine at position 128.

The polypeptide may be an "immature" or "full-length" AmyDSM 90-related polypeptide that includes a signal sequence, or an AmyDSM 90-related polypeptide that lacks the mature form of the signal sequence. An exemplary immature form of AmyDSM90 polypeptide has the amino acid sequence of SEQ ID NO: 2, and an exemplary mature form of an AmyDSM90 polypeptide has the amino acid sequence of SEQ ID NO: 1. The mature form of the polypeptide is mostly used in cleaning compositions. The polypeptide may be a truncated form of an AmyDSM 90-related polypeptide that lacks the N-terminus or C-terminus of the mature form, or a fragment of an AmyDSM 90-related polypeptide that still retains at least a portion of the alpha-amylase activity characteristic of the parent AmyDSM 90-related polypeptide.

As noted above, the polypeptides include variant polypeptides, such as those that include artificial deletions, insertions, and substitutions. Exemplary deletions are of residues R179 and/or G180. An exemplary substitution is a residue M200, e.g., a M200L substitution. The R179-G180 deletion and M200 substitution can also be combined in a single polypeptide. Other exemplary substitutions are conservative amino acid substitutions, such as those listed in the following table.

As mentioned, preferred polypeptides still retain alpha-amylase activity, but may have altered biochemical properties relative to the naturally occurring parent polypeptide. In some embodiments, the parent polypeptide is SEQ ID NO: 1.

The polypeptide can also be a chimeric polypeptide comprising at least a portion of an AmyDSM 90-related polypeptide and at least a portion of a second polypeptide. The second polypeptide may for example be a second amylase, a heterologous signal sequence, an epitope allowing tracking or purification, etc. Exemplary heterologous signal sequences are from Bacillus subtilis amylase (AmyE) or AprE and Streptomyces CelA.

Another aspect of the compositions and methods of the invention are nucleic acids encoding AmyDSM 90-related polypeptides. The nucleic acid may encode AmyDSM90, an amylase having a specified degree of identity to AmyDSM90, a variant of AmyDSM90 that includes an artificial substitution, insertion, or deletion, or a chimera thereof. In one example, the nucleic acid encodes an amylase that has 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 AmyDSM90 polypeptide (e.g., the polypeptide of SEQ ID NO: 1). The polypeptide may include at least one of the amino acid sequence features described above. It is understood that due to the degeneracy of the genetic code, multiple nucleic acids may encode the same polypeptide.

The nucleic acid may also be related to an exemplary polynucleotide encoding an AmyDSM 90-related polypeptide such as SEQ ID NO: 7 have a specified degree of homology. In one example, the nucleic acid is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identical to an exemplary sequence. In another example, the nucleic acid hybridizes to the exemplary sequence under stringent or very stringent conditions.

The nucleic acid may encode a "full-length" ("FL" or "FL") AmyDSM 90-related polypeptide including a signal sequence, an only mature form of AmyDSM 90-related polypeptide lacking a signal sequence, a truncated form of AmyDSM 90-related polypeptide lacking the N-terminus or C-terminus of the mature form, or a fragment thereof, that still retains at least a portion of the alpha-amylase activity characteristic of AmyDSM 90-related polypeptides.

The nucleic acid encoding the AmyDSM 90-related polypeptide may be operably linked to a variety of promoters and regulatory genes in vectors suitable for expression of the AmyDSM 90-related polypeptide in a host cell. Exemplary promoters are the Bacillus subtilis Amy E promoter and the AprE promoter, and the Streptomyces CelA promoter. Such nucleic acids may also be linked to other coding sequences, for example intended to encode a chimeric polypeptide.

3.Method for producing and purifying proteins

One aspect of the compositions and methods of the invention is that the AmyDSM 90-related polypeptides may be expressed as secreted polypeptides. Notably, based on the annotations in the Genbank entry, orthologous alpha-amylases from Bacillus anthracis AAT32659(SEQ ID NO: 4), Bacillus thuringiensis AAT60457(SEQ ID NO: 5) and Bacillus cereus AAP10417(SEQ ID NO: 6) were considered to be cellular polypeptides to date. For industrial and commercial processes, cytosolic amylases are considered to be of lower value because their production and recovery is more complex. Thus, it was found that AmyDSM 90-related polypeptides (including these orthologous alpha-amylases) are secreted, greatly facilitating their isolation and purification, and enabling their large-scale use in industrial and commercial processes.

Methods for producing and purifying proteins secreted from bacillus into culture media are known in the art, and suitable host cells for producing alpha-amylase are also known in the art. Exemplary methods for producing alpha-amylase are disclosed below.

3.1Materials and methods for producing alpha-amylase

AmyDSM 90-related polypeptides can be expressed enzymatically using an expression vector that typically includes regulatory 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 in recombinant DNA procedures, and the choice of vector will often depend on the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a phage or an extrachromosomal element, a minichromosome, 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 can also be amplified to produce multiple copies of the gene in the chromosome by using an amplification-type construct driven by antibiotic selection or other selection pressure (e.g., an essential regulatory gene) or by complementation through the 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 which 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 transcription of a DNA sequence encoding an AmyDSM 90-related polypeptide, particularly in a bacterial host, are the promoter of the e.coli lac operon, the promoter of the streptomyces coelicolor agarase gene dagA or celA, the promoter of the bacillus licheniformis alpha-amylase gene (amyL), the promoter of the bacillus stearothermophilus maltogenic amylase gene (amyM), the promoter of the bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the bacillus subtilis xylA and xylB genes, and the like. Examples of useful promoters for transcription in a fungal host are those derived from the genes encoding Aspergillus oryzae (A.oryzae) TAKA amylase, Rhizomucor miehei (Rhizomucor miehei) aspartic proteinase, Aspergillus niger (A.niger) neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger glucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae trisaccharide phosphate isomerase, or Aspergillus nidulans (A.nidulans) acetamidase. When a gene encoding an AmyDSM 90-related polypeptide is expressed in a bacterial species such as escherichia coli, an appropriate promoter may be selected, for example, from phage promoters including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for expression in yeast species include, but are not limited to, the Saccharomyces cerevisiae (Saccharomyces cerevisiae) Gal 1 and Gal 10 promoters and the Pichia pastoris (Pichia pastoris) AOX1 or AOX2 promoter. For expression in trichoderma reesei (trichoderma reesei), the CBHII (cellobiohydrolase II) promoter may be used.

The expression vector may also comprise a suitable transcription terminator and (in eukaryotes) a polyadenylation sequence operably linked to the DNA sequence encoding the AmyDSM 90-related polypeptide. The termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may also 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 pIJ 702.

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 which confers antibiotic resistance (e.g., penicillin, kanamycin, chloramphenicol, or tetracycline resistance). In addition, the vector may contain Aspergillus selection markers such as amdS, argB, niaD and xxsC for hygromycin resistance, or selection may be achieved by co-transformation methods known in the art. See, for example, international PCT application WO 91/17243.

As noted above, while intracellular expression or solid state fermentation may be advantageous in some aspects, for example, when certain bacteria or fungi are used as host cells, one aspect of the compositions and methods contemplates the expression of AmyDSM 90-related polypeptides into the culture medium.

Typically, full-length or immature AmyDSM 90-related polypeptides include a signal sequence at the hydrogen terminus that allows secretion into the culture medium. The signal peptide may be replaced by a different sequence as required, conveniently by replacing the DNA sequence encoding the respective signal polypeptide.

Expression vectors generally include components of the cloning vector, e.g., elements that permit autonomous replication of the vector in a selected host organism and one or more phenotypically detectable markers for selection purposes. Expression vectors typically comprise regulatory nucleotide sequences, such as a promoter, an operator, a ribosome binding site, a translation initiation signal, and optionally a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence encoding an amino acid sequence, wherein said amino acid sequence is capable of targeting the alpha-amylase variant to an organelle of the host cell, such as the peroxisome, or to a specific host cell compartment. Such targeting sequences include, but are not limited to, the sequence SKL. For expression under the direction of the control sequences, the nucleic acid sequence of the alpha-amylase variant is operatively linked to the control sequences in a manner appropriate for expression. A portion of an exemplary carrier is depicted in fig. 3.

The DNA constructs used to ligate the AmyDSM 90-related polypeptides, promoters, terminators and other elements, respectively, and methods used to insert them into suitable vectors containing the information necessary for replication are well known to those skilled in the art (see, e.g., Sambrook et al, Molecularcolong: A LABORATORY MANUAL, 2 nd edition, Cold Spring Harbor, 1989, and 3 rd edition, 2001).

An isolated cell comprising a DNA construct or expression vector is advantageously used as a host cell in the recombinant production of an AmyDSM 90-related polypeptide. The cell may be transformed with a DNA construct encoding the enzyme, conveniently by integration of the DNA construct (in one or more copies) in the host chromosome. Such integration is generally considered advantageous because the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct into the host chromosome may be carried out according to conventional methods, for example by homologous or heterologous recombination. Alternatively, the cells may be transformed with an expression vector as described above in connection with different types of host cells.

Examples of suitable bacterial host organisms are gram-positive bacterial species, such as the Bacillaceae family, including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus (Bacillus lentus), Bacillus brevis (Bacillus brevis), Geobacillus stearothermophilus (where Geobacillus geophilus was previously referred to as Bacillus (Geobacillus stearothermophilus), Bacillus alkalophilus (Bacillus alkalophilus), Bacillus amyloliquefaciens (Bacillus coaggulans), Bacillus coagulans (Bacillus coaggulans), Bacillus lautus (Bacillus lautus), Bacillus megaterium and Bacillus thuringiensis; streptomyces species, such as Streptomyces murinus (Streptomyces murinus); lactic acid bacterial species, including Lactococcus species (Lactococcus sp.), such as Lactococcus lactis (Lactococcus lactis); lactobacillus species (Lactobacillus sp.), including Lactobacillus reuteri (Lactobacillus reuteri); leuconostoc species (Leuconostoc sp.); pediococcus sp and Streptococcus sp. Alternatively, strains of gram-negative bacterial species belonging to the family Enterobacteriaceae (including escherichia coli) or to the family pseudomonas (pseudomonas adaceae) may be selected as host organisms.

Suitable yeast host organisms may be selected from biotechnologically relevant yeast species, such as, but not limited to, Saccharomyces species, such as Pichia species (Pichia sp.), Hansenula species (Hansenula sp.) or Kluyveromyces species, yarrowia species, Schizosaccharomyces species or Saccharomyces species (Saccharomyces), including Saccharomyces cerevisiae, or species belonging to the genus Schizosaccharomyces, such as Schizosaccharomyces pombe (S.pombe) species. Strains of the methylotrophic yeast species Pichia pastoris can be used as host organisms. Alternatively, the host organism may be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus (Aspergillus), such as Aspergillus niger (Aspergillus niger), Aspergillus oryzae (Aspergillus oryzae), Aspergillus tubingensis (Aspergillus oryzae), Aspergillus awamori (Aspergillus awamori), or Aspergillus nidulans (Aspergillus nidulan). Alternatively, strains of Fusarium (Fusarium) species (e.g., Fusarium oxysporum) or rhizomucor species (e.g., rhizomucor miehei) may be used as host organisms. Other suitable strains include Thermomyces (Thermomyces) and Mucor species. In addition, Trichoderma reesei can be used as a host organism. Suitable methods for transforming an aspergillus host cell include, for example, the methods described in EP 238023.

In yet another aspect, a method of producing an AmyDSM 90-related polypeptide is provided, the method comprising culturing a host cell as described above under conditions conducive to production of the enzyme and recovering the enzyme from the cell and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for cultivating the host cell in question and obtaining expression of the AmyDSM 90-related polypeptide. 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, the enzyme secreted from the host cell is used in a complete culture broth preparation. In the method of the invention, the preparation of a complete fermentation broth, which has been used by the recombinant microorganism, can be achieved using any cultivation method known in the art which results in the expression of an alpha-amylase. Thus, fermentation is understood to include shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid state fermentations) in laboratory or industrial fermentors 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 the unfractionated content of fermentation material including culture medium, extracellular proteins (e.g., enzymes) and cellular biomass. It is to be understood that the term "spent whole fermentation broth" also includes a biomass of cells that have been lysed or permeabilized using methods well known in the art.

The enzyme secreted from the host cell may be conveniently recovered from the culture medium by well-known methods including separation of the cells from the medium by centrifugation or filtration, and precipitation of the proteinaceous components by means of a salt, such as ammonium sulphate, followed by the use of chromatographic methods such as ion exchange chromatography, affinity chromatography and the like.

One aspect contemplates that the polynucleotide in the vector is operably linked to a control sequence capable of causing expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The regulatory sequence may be modified, for example by the addition of other transcriptional regulatory elements, to make the level of transcription directed by the regulatory sequence more responsive to the transcriptional regulator. The control sequence may in particular comprise a promoter.

The host cell may be cultured under suitable conditions that allow for the expression of an AmyDSM 90-related polypeptide. The expression of the enzymes may be constitutive, so that they are produced continuously, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, it is possible, when desired, to effect the desired expression, for example by adding an inducing substance such as dexamethasone,IPTG or sepharose to the medium, protein production is initiated. The polypeptides may also be in vitro in cell-free systems (e.g., TNT)^TM(Promega) rabbit reticulocyte system).

The host expressing the AmyDSM 90-related polypeptide may also be cultured under aerobic conditions in a medium suitable for the host. Shaking may be provided or a combination of stirring and aeration may be provided while production is performed at a suitable temperature for the host, such as from about 25 ℃ to about 75 ℃ (e.g., 30 ℃ to 45 ℃), depending on the host and the need to produce the desired alpha-amylase variant. The culture may be carried out for about 12 hours to about 100 hours or more (and any time value in between, e.g., from 24 hours to 72 hours generally, the pH of the culture broth is from about 5.5 to about 8.0, again depending on the culture conditions required for the host cell to produce the AmyDSM 90-related polypeptide.

3.2Materials and methods for protein purification

Fermentation, isolation and concentration techniques are well known in the art and conventional methods can be used to prepare solutions containing concentrated AmyDSM 90-related polypeptides.

After fermentation, a fermentation broth is obtained, from which the microbial cells and various suspended solids (including residual fermentation feedstock) are removed by conventional separation techniques, with the aim of obtaining an amylase solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration (ultrafiltration), post-centrifugation ultrafiltration, extraction, chromatography, or the like is generally used.

It is desirable to concentrate the solution containing AmyDSM 90-related polypeptide to optimize recovery. The use of a non-concentrated solution requires an increased incubation time 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 can be achieved by any of the techniques discussed herein. Exemplary methods of purification include, but are not limited to, rotary vacuum filtration and/or ultrafiltration.

The enzyme solution is concentrated to a concentrated enzyme solution until the enzyme activity of the solution containing the concentrated AmyDSM 90-related polypeptide is at a desired level.

Concentration may be carried out using, for example, a precipitating agent such as a metal halide precipitating agent. Metal halide precipitants 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 precipitant sodium chloride may also be used as a preservative,

the metal halide precipitation agent is used in an amount effective to precipitate the AmyDSM 90-related polypeptide. It will be readily apparent to one of ordinary skill in the art, after routine experimentation, that at least an effective and optimal amount of metal halide effective to cause precipitation of the enzyme, as well as the precipitation conditions (including incubation time, pH, temperature, enzyme concentration, etc.) for maximum recovery, will be selected.

Generally, at least about 5% w/v (weight/volume) to about 25% w/v metal halide is added to the concentrated enzyme solution, and typically at least 8% w/v metal halide. Generally, no more than about 25% w/v metal halide is added to the concentrated enzyme solution, and typically no more than about 20% w/v metal halide. The optimum concentration of the metal halide precipitant depends inter alia on the nature of the particular AmyDSM 90-related polypeptide and on its concentration in the concentrated enzyme solution.

Another alternative to achieve enzyme precipitation is the use of 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 the organic compound precipitant may be performed before, simultaneously with, or after the addition of the metal halide precipitant, and the addition of the two precipitants (organic compound and metal halide) may be performed sequentially or simultaneously.

Generally, the organic precipitating agent is selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid (e.g., sodium or potassium salts) and linear or branched alkyl 4-hydroxybenzoates in which the alkyl group contains 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitating agent may be, for example, a linear or branched alkyl 4-hydroxybenzoate, wherein the alkyl group contains 1 to 10 carbon atoms, and a blend of two or more of these organic compounds. Exemplary organic compounds are linear alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains 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 esters of 4-hydroxybenzoic acid, ethyl esters of 4-hydroxybenzoic acid and blends of two or more of these organic compounds may also be used. Additional organic compounds also include, but are not limited to, methyl 4-hydroxybenzoate (named methyl paraben), propyl 4-hydroxybenzoate (named propyl paraben), both of which are also amylase preservatives. For further description, see, e.g., U.S. patent No. 5,281,526.

The addition of the organic compound precipitant provides the advantage of high flexibility of the precipitation conditions in terms of pH, temperature, AmyDSM 90-related polypeptide concentration, precipitant concentration, and incubation time.

The organic compound precipitant is used in an amount effective to improve precipitation of the enzyme by the metal halide precipitant. After routine experimentation, it will be readily apparent to one of ordinary skill in the art, given the teachings of this disclosure, that at least an effective and optimal amount of organic compound precipitant, as well as precipitation conditions for maximum recovery, including incubation time, pH, temperature, enzyme concentration, and the like, can be selected.

Generally, at least about 0.01% w/v of the organic compound precipitating agent is added to the concentrated enzyme solution, and typically at least about 0.02% w/v of the organic compound precipitating agent. Generally, no more than about 0.3% w/v of organic compound precipitant is added to the concentrated enzyme solution, and typically no more than about 0.2% w/v of organic compound precipitant.

The concentrated AmyDSM 90-related polypeptide solution containing the metal halide precipitant and the organic compound precipitant can be adjusted to a pH that will necessarily depend on the enzyme to be purified. Typically, the pH is adjusted to a level near the isoelectric point of the amylase. For example, the pH can be adjusted in a range from about 2.5pH units below the isoelectric point (pI) to about 2.5pH units above the isoelectric point.

The incubation time required to obtain a purified enzyme precipitate depends on the nature of the specific enzyme, the enzyme concentration, the specific precipitating agent and its (their) concentration. Typically, the time effective to precipitate the enzyme is between about 1 hour to about 30 hours; typically, it does not exceed about 25 hours. In the presence of the organic compound precipitating agent, the incubation time can still be reduced to less than about 10 hours, and in most cases even less than about 6 hours.

Typically, the temperature during incubation is between about 4 ℃ and about 50 ℃. Typically, the process is carried out at between about 10 ℃ and about 45 ℃ (e.g., between about 20 ℃ and about 40 ℃). The optimum temperature for inducing precipitation varies depending on the solution conditions and the enzyme or precipitating agent used.

By stirring the solution comprising the enzyme, the added metal halide and the added organic compound, the overall recovery of the purified enzyme precipitate and the efficiency of carrying out the process are improved. The stirring step is carried out both during the addition of the metal halide and the organic compound and during the subsequent incubation time. Suitable methods of agitation include mechanical agitation or shaking, vigorous aeration or any similar technique.

After the incubation period, the purified enzyme is then separated from the dissociated pigments and other impurities and collected by conventional separation techniques (e.g., filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, pressure filtration, transmembrane microfiltration, cross-flow membrane microfiltration, etc.). Further purification of the purified enzyme precipitate may be obtained by washing the precipitate with water. For example, the purified enzyme precipitate is washed with water containing a metal halide precipitant or with water containing a metal halide and an organic compound precipitant.

During fermentation, AmyDSM 90-related polypeptides accumulate in the culture broth. To isolate and purify the desired AmyDSM 90-related polypeptide, 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 culture broth is salted out with ammonium sulfate at a saturation of about 70%; the 70% saturated precipitated fraction is then dissolved in buffer and applied to a column such as a Sephadex G-100 column and eluted to recover the enzymatically active fraction. For further purification, conventional methods such as ion exchange chromatography and the like can be used.

Purified enzymes are used in laundry and cleaning applications. For example, they can be used in laundry detergents and stain removers. They can be prepared as end products either as liquids (solutions and slurries) or solids (granules and powders).

More specific examples of purification are in j.sumitani et al, "novel starch binding domain: direct repeat motifs in the C-terminal region of alpha-amylase No.195 from Bacillus species contribute to starch binding and raw starch degradation (New type of starch-binding domain: the direct repeat motif the C-terminal region of Bacillus sp.no.195 alpha-amylase binding and raw starch degradation), "biochem.J.350: 477-484(2000) and briefly summarized here. The enzyme obtained from the culture supernatant of 4 liters of Streptomyces lividans TK24 was saturated with (NH) 80%₄)₂SO₄And (6) processing. The pellet was recovered by centrifugation at 10,000x g (20 min and 4 ℃) and redissolved in a medium containing 5mM CaCl₂In 20mM Tris/HCl buffer (pH 7.0). The dissolved pellet was then dialyzed against the same buffer. The dialyzed sample was then applied to a Sephacryl S-200 column, which had previously been loaded with 20mM Tris/HCl buffer (pH 7.0), 5mM CaCl₂Equilibrated and eluted with the same buffer at a linear flow rate of 7 mL/hour. Fractions from the column were collected and their activity was assessed as by enzyme assay and SDS-PAGE. The protein was further purified as follows. Toyopearl HW55 column (Tosoh Bio)science, Montgomeryville, PA; cat. No. 19812) with a solution containing 5mM CaCl₂And 1.5M (NH)₄)₂SO₄Equilibrated in 20mM Tris/HCl buffer (pH 7.0). The enzyme was used in a medium containing 5mM CaCl₂20mM Tris/HCl buffer, 1.5 to 0M (NH) at pH 7.0₄)₂SO₄Elution with a linear gradient. The active fractions were collected and saturated with 80% (NH)₄)₂SO₄The enzyme is precipitated. The precipitate was recovered, redissolved 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 loaded with 5mM CaCl₂Is equilibrated at a flow rate of 60 mL/hour in 20mM Tris/HCl buffer (pH 7.0). Active fractions were collected and added to 1.5M (NH)₄)₂SO₄. The active enzyme fraction was re-chromatographed on a Toyopearl HW55 column as before to yield a homogeneous enzyme as determined by SDS-PAGE. For a general discussion of this approach and its variants, see j.sumitani et al, "novel starch binding domains: direct repetitive motifs in the C-terminal region of alpha-amylase No.195 from Bacillus species contribute to starch binding and raw starch degradation (New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp.no.195 alpha-amylase constructs to starch binding and raw starch degradation), "biochem.J.350: 477-484(2000).

For production scale recovery, the AmyDSM 90-related polypeptide can be partially purified by removing cells by flocculation with a polymer as generally described above. Alternatively, using available membranes and equipment, the enzyme may be purified by microfiltration followed by concentration by ultrafiltration. However, for some applications, the enzyme need not be purified, and the complete broth culture can be lysed and used without further processing. The enzyme may be subsequently processed, for example into granules.

4.Cleaning composition

One aspect of the compositions and methods of the present invention is a cleaning composition comprising an AmyDSM 90-related polypeptide as a component. The AmyDSM 90-related polypeptides can be used as components in detergent compositions for hand washing, laundry washing, dish washing, and other hard surface cleaning. Preferably, the AmyDSM 90-related polypeptide is incorporated into the detergent at or near the concentrations at which amylases are conventionally used in detergents. For example, the AmyDSM 90-related polypeptide may be added in an amount corresponding to 0.00001-1mg (calculated as pure enzyme protein) of alpha-amylase per liter of wash/dish wash. Exemplary formulations are provided herein, as exemplified below:

4.1laundry detergent composition

The AmyDSM 90-related polypeptide may typically be a component of a detergent composition, either as the only enzyme or in conjunction with other enzymes, including other amylolytic enzymes. As such, it may be included in the detergent composition in the form of a non-dusting granule, a stabilized liquid, or a protected enzyme. Non-dusting granules may be produced, for example, 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 (polyethylene glycol, PEG) having an average molar weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which 15 to 80 ethylene oxide units are present; a fatty alcohol; a fatty acid; and fatty acids monoacylglycerols and diacylglycerols and triacylglycerols. Examples of film-forming coating materials suitable for use in fluid bed techniques are given in british patent No. 1483591. The liquid enzyme preparation may be stabilized according to established methods, for example by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid. Other enzyme stabilizers are known in the art. The protected enzymes may be prepared according to the methods disclosed in, for example, EP 238,216. Polyols have long been considered as protein stabilizers and to improve protein solubility. See J.K. Kaushik et al, "what is a trehalose an experimental protein scaffold? (why trehalose is an unexpected protein stabilizer: 26458-65(2003) and the references cited therein; and monica. conti et al, "capaclary isoelectric housing: the protein of protein solubility (capillary isoelectric focusing: problem of protein solubility), "J.Chromograph A757: 237-245(1997).

The detergent composition may be in any useful form, for example, as a powder, granule, paste or liquid. Liquid detergents may be aqueous and typically contain up to about 70% water and 0 to about 30% organic solvent. It may also be in the form of a dense 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 typically contain from 0% to about 50% anionic surfactant, such as linear alkyl benzene sulphonate (LAS); alpha-olefmic sulfonates (AOS); alkyl sulfates (fatty Alcohol Sulfates) (AS); alcohol ethoxylated sulfates (AEOS or AES); secondary Alkyl Sulfonates (SAS); alpha-sulfo fatty acid methyl ester; alkyl or alkenyl succinic acids; or a soap. The composition may contain from 0% to about 40% of a non-ionic surfactant such as an alcohol ethoxylate (AEO or AE), a carboxylated alcohol ethoxylate, a nonylphenol ethoxylate, an alkylpolyglycoside, an alkyldimethyamine oxide, an ethoxylated fatty acid monoethanolamide, a fatty acid monoethanolamide, or a polyhydroxyalkyl fatty acid amide (e.g. as described 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 from 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 alkenyl succinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst). The detergent may also be non-builder, i.e. substantially free of detergent builder. The enzyme may be used in any composition that is compatible with enzyme stability. The enzymes can generally be protected from harmful components by known encapsulation forms, for example by granulation or isolation in hydrogels. Enzymes, with or without a starch binding domain, and in particular alpha-amylases, such as the AmyDSM90 molecule, can be used in a variety of compositions, including laundry and dishwashing applications, surface cleaners, and in compositions for producing ethanol from starch or biomass.

The detergent may comprise one or more polymers. Examples include carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), and polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system, which may comprise H₂O₂Sources such as perborate or percarbonate which may be combined with peracid-forming bleach activators such as Tetraacetylethylenediamine (TAED) or Nonanoyloxybenzenesulfonate (NOBS). Alternatively, the bleaching system may comprise peroxyacids (e.g. of the amide, imide or sulfone type). The bleaching system may also be an enzymatic bleaching system, for example, a perhydrolase enzyme, such as the one described in international PCT application WO 2005/056783.

The enzymes of the detergent composition may be stabilized using conventional stabilizers, for example polyols such as propylene glycol or glycerol; a sugar or sugar alcohol; lactic acid; boric acid or boric acid derivatives, such as aromatic borate esters; and the compositions may be formulated as described in WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as fabric softeners, including clays, sudsing agents, suds suppressors, anti-corrosion agents, soil suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibitors, optical brighteners or perfumes.

The pH (measured in aqueous solution at the use concentration) is typically neutral or basic, e.g., a pH of about 7.0 to about 11.0.

Particular forms of detergent compositions comprising AmyDSM 90-related polypeptides may be formulated to include:

(1) a detergent composition formulated as granules having a bulk density of at least 600g/L comprising from about 7% to about 12% linear alkylbenzene sulfonate (calculated as acid); about 1% to about 4% alcohol ethoxy sulfate (e.g., C)_12-18Alcohols, 1-2 Ethylene Oxide (EO)) or alkyl sulfates (e.g. C)_16-18) (ii) a About 5% to about 9% alcohol ethoxylate (e.g., C)_14-15Alcohol, 7 EO); about 14 to about 20% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 2% to about 6% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 15% to about 22% of a zeolite (e.g., NaAlSiO @₄) (ii) a 0% to about 6% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 0% to about 15% sodium citrate/citric acid (e.g., C)₆H₅Na₃O₇/C₆H₈O₇) (ii) a About 11% to about 18% sodium perborate (e.g., NaBO)₃H₂O); about 2% to about 6% TAED; 0% to about 2% carboxymethylcellulose (CMC); 0-3% of a polymer (e.g. maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% protein (calculated as pure enzyme); and from 0% to about 5% of minor ingredients (e.g., suds suppressors, perfumes, brighteners, photobleaches).

(2) A detergent composition formulated as granules having a bulk density of at least 600g/L comprising from about 6% to about 11% linear alkylbenzene sulfonate (calculated as acid); about 1% to about 3% alcohol ethoxy sulfate (e.g., C)_12-18Alcohols, 1-2EO) or alkyl sulfates (e.g. C)_16-18) (ii) a About 5% to about 9% alcohol ethoxylate (e.g., C)_14-15Alcohol, 7 EO); about 15% to about 21% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 1% to about 4% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 24% to about 34% of a zeolite (e.g., NaAlSiO ™)₄) (ii) a 4% to about 10% sodium sulfate (e.g., Na)₂SO₄) (ii) a 0% to about 15% sodium citrate/citric acid (e.g. C)₆H₅Na₃O₇/C₆H₈O₇) (ii) a 0% to about 2% carboxymethylcellulose (CMC); 1-6% of a polymer (e.g. maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0-5% of minor ingredients (such as suds suppressors, perfume).

(3) A detergent composition formulated as granules having a bulk density of at least 600g/L comprising from about 5% to about 9% linear alkylbenzene sulfonate (calculated as acid); about 7% to about 14% alcohol ethoxylate (e.g., C)_12-15Alcohol, 7 EO); from about 1 to about 3% soap as a fatty acid; about 10% to about 17% sodium carbonate (as Na)₂CO₃) (ii) a About 3% to about 9% of a soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 23% to about 33% of zeolite (as NaA1 SiO)₄) (ii) a 0% to about 4% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 8% to about 16% sodium perborate (e.g., NaBO)₃H₂O); about 2% to about 8% TAED; 0% to about 1% of a phosphonate (e.g., EDTMPA); 0% to about 2% carboxymethylcellulose (CMC); 0-3% polymer (e.g., maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0-5% of minor ingredients (e.g. suds suppressors, perfume, optical brighteners).

4) A detergent composition formulated as granules having a bulk density of at least 600g/L comprising from about 8% to about 12% linear alkylbenzene sulfonate (calculated as acid); about 10% to about 25% alcohol ethoxylate (e.g., C)_12-15Alcohol, 7 EO); about 14% to about 22% sodium carbonate (as Na)₂CO₃) (ii) a About 1% to about 5% soluble silicate (e.g., Na)₂O、2SiO₂) (ii) a About 25% to about 35% of a zeolite (e.g., NaA1 SiO)₄) (ii) a 0% to about 10% sodium sulfate (e.g., Na)₂SO₄) (ii) a 0% to about 2% carboxymethylcellulose (CMC); 1-3% polymer (e.g., maleic/acrylic acid copolymer, PVP, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., suds suppressors, perfume).

An aqueous liquid detergent composition comprising from about 15% to about 21% linear alkylbenzene sulfonate (calculated as acid); about 12% to about 18% alcohol ethoxylate (e.g., C)_12-15Alcohol, 7EO or C_12-15Alcohol, 5 EO); from about 3% to about 13% soap as a fatty acid (e.g., oleic acid); 0% to about 13% alkenyl succinic acid (C)_12-14) (ii) a About 8% to about 18% aminoethanol; about 2% to about 8% citric acid; 0% to about 3% of a phosphonate; 0% to about 3% of a polymer (e.g., PVP, PEG); 0% to about 2% of a borate (e.g., B)₄O₇) (ii) a 0% to about 3% ethanol; about 8% to about 14% propylene glycol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., dispersants, suds suppressors, perfumes, optical brighteners).

An aqueous structured liquid detergent composition comprising from about 15% to about 21% linear alkylbenzene sulfonate (calculated as acid); 3-9% alcohol ethoxylate (e.g., C)_12-15Alcohol, 7EO or C_12-15Alcohol, 5 EO); from about 3% to about 10% soap as a fatty acid (e.g., oleic acid); about 14% to about 22% zeolite (as NaA1 SiO)₄) (ii) a From about 9% to about 18% potassium citrate; 0% to about 2% of a borate (e.g., B)₄O₇) (ii) a 0% to about 2% carboxymethylcellulose (CMC); 0% to about 3% of a polymer (e.g., PEG, PVP); 0% to about 3% of an anchoring polymer, for example, lauryl methacrylate/acrylic acid copolymer; molar ratio 25: 1, MW 3800); 0% to about 5% glycerin; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., dispersants, suds suppressors, perfumes, optical brighteners).

7) A detergent composition formulated as granules having a bulk density of at least 600g/L comprising from about 5% to about 10% fatty alcohol sulfate; from about 3% to about 9% ethoxylated fatty acid monoethanolamide; 0-3% soap as a fatty acid; about 5% to about 10% sodium carbonate(e.g., Na)₂CO₃) (ii) a About 1% to about 4% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 20% to about 40% of a zeolite (e.g., NaA1 SiO)₄) (ii) a About 2% to about 8% sodium sulfate (e.g., Na)₂SO₄) (ii) a Sodium perborate (e.g., NaBO) from about 12% to about 18%₃H₂O); about 2% to about 7% TAED; about 1% to about 5% of a polymer (e.g., maleic/acrylic acid copolymer, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., optical brighteners, suds suppressors, perfumes).

8) A detergent composition formulated as granules comprising from about 8% to about 14% linear alkylbenzene sulfonate (calculated as acid); from about 5% to about 11% ethoxylated fatty acid monoethanolamide; from 0% to about 3% soap as a fatty acid; about 4% to about 10% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 1% to about 4% soluble silicate (Na)₂O，2SiO₂) (ii) a About 30% to about 50% of a zeolite (e.g., NaA1 SiO)₄) (ii) a About 3% to about 11% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 5% to about 12% sodium citrate (e.g., C)₆H₅Na₃O₇) (ii) a About 1% to about 5% of a polymer (e.g., PVP, maleic/acrylic acid copolymer, PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., suds suppressors, perfume).

9) A detergent composition formulated as granules comprising from about 6% to about 12% linear alkylbenzene sulfonate (calculated as acid); from about 1% to about 4% of a nonionic surfactant; from about 2% to about 6% soap as a fatty acid; about 14% to about 22% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 18% to about 32% of a zeolite (e.g., NaA1 SiO)₄) (ii) a About 5% to about 20% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 3% to about 8% sodium citrate (e.g., C)₆H₅Na₃O₇) (ii) a About 4% to about 9% sodium perborate (e.g., NaBO)₃H₂O); about 1% to about 5% of a bleach activator (e.g., NOBS or TAED); 0% to about 2% carboxymethylcellulose (CMC); about 1% to about 5% of a polymer (e.g., polycarboxylate or PEG); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% of minor ingredients (e.g., optical brighteners, perfumes).

10) An aqueous liquid detergent composition comprising from about 15% to about 23% linear alkylbenzene sulfonate (calculated as acid); from about 8% to about 15% alcohol ethoxylated sulfate (e.g., C)_12-15Alcohol, 2-3 EO); about 3% to about 9% alcohol ethoxylate (e.g., C)_12-15Alcohol, 7EO, or C_12-15Alcohol, 5 EO); 0% to about 3% soap as a fatty acid (e.g., lauric acid); about 1% to about 5% aminoethanol; from about 5% to about 10% sodium citrate; about 2% to about 6% of a hydrotrope (e.g., sodium toluene sulfonate); 0% to about 2% of a borate (e.g., B)₄O₇) (ii) a 0% to about 1% carboxymethylcellulose; about 1% to about 3% ethanol; about 2% to about 5% propylene glycol; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% of minor ingredients (e.g., polymers, dispersants, perfumes, optical brighteners).

11) An aqueous liquid detergent composition comprising from about 20% to about 32% linear alkylbenzene sulfonate (calculated as acid); 6-12% alcohol ethoxylate (e.g., C)_12-15Alcohol, 7EO, or C_12-15Alcohol, 5 EO); about 2% to about 6% hydrino ethanol; about 8% to about 14% citric acid; about 1% to about 3% of a borate (e.g., B)₄O₇) (ii) a 0% to about 3% of a polymer (e.g., maleic/acrylic acid copolymer, anchoring polymer, e.g., lauryl methacrylate/acrylic acid copolymer); about 3% to about 8% glycerin; 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% minor ingredients (e.g., hydrotropes, dispersants, perfumes, optical brighteners).

12) A detergent composition formulated as a granule having a bulk density of at least 600g/L comprising from about 25% to about 40% of an anionic surfactant (linear alkylbenzene sulphonate,Alkyl sulfates, alpha-olefinsulfonates, alpha-sulfo fatty acid methyl esters, alkyl sulfonates, soaps); from about 1% to about 10% of a nonionic surfactant (e.g., an alcohol ethoxylate); about 8% to about 25% sodium carbonate (e.g., Na)₂CO₃) (ii) a About 5% to about 15% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a 0% to about 5% sodium sulfate (e.g., Na)₂SO₄) (ii) a About 15% to about 28% of zeolite (NaA1 SiO)₄) (ii) a Sodium perborate (e.g., NaBO) from 0% to about 20%₃·4H₂O); from about 0% to about 5% bleach activator (TAED or NOBS); 0.0001-0.1% enzyme (calculated as pure enzyme protein); 0-3% of minor ingredients (e.g. perfumes, optical brighteners).

13) Detergent composition as described in the above compositions 1) to 12), wherein all or part of the linear alkylbenzene sulfonate is substituted by (C)₁₂-C₁₈) Alkyl sulfate substitution.

14) A detergent composition formulated as a granule having a bulk density of at least 600g/L comprising from about 9% to about 15% (C)₁₂-C₁₈) An alkyl sulfate; from about 3% to about 6% alcohol ethoxylate; from about 1% to about 5% of a polyhydroxy alkyl fatty acid amide; about 10% to about 20% of a zeolite (e.g., NaA1 SiO)₄) (ii) a From about 10% to about 20% of a layered disilicate (e.g., SK56 from Hoechst); about 3% to about 12% sodium carbonate (e.g., Na)₂CO₃) (ii) a 0% to about 6% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a From about 4% to about 8% sodium citrate; about 13% to about 22% sodium perborate; about 3% to about 8% TAED; 0% to about 5% of a polymer (e.g., polycarboxylate and PVP); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-5% of minor ingredients (e.g., optical brighteners, photobleaches, perfumes, suds suppressors).

15) A detergent composition formulated as a granule having a bulk density of at least 600g/L comprising from about 4% to about 8% (C)₁₂-C₁₈) An alkyl sulfate; about 11% to about 15% of an alcoholAn ethoxylate; from about 1% to about 4% soap; from about 35% to about 45% zeolite MAP or zeolite a; about 2% to about 8% sodium carbonate (as Na)₂CO₃) (ii) a 0% to about 4% soluble silicate (e.g., Na)₂O，2SiO₂) (ii) a About 13% to about 22% sodium perborate; 1-8% TAED; 0% to about 3% carboxymethylcellulose (CMC); 0% to about 3% of a polymer (e.g., polycarboxylate and PVP); 0.0001-0.1% enzyme (calculated as pure enzyme protein); and 0-3% minor ingredients (e.g., optical brighteners, phosphonates, perfumes).

16) Detergent formulations as described in 1) to 15) above, which contain a stable or encapsulated peracid as an additional component or as a replacement for already specified bleaching systems.

17) Detergent compositions as described in 1), 3), 7), 9) and 12) above, wherein the perborate is replaced by percarbonate.

18) A detergent composition as described in 1), 3), 7), 9), 12), 14) and 15) above, additionally containing a manganese catalyst. Such a manganese catalyst is, for example, "high-efficiency manganese catalysts for low-temperature bleaching (effective catalyst catalysts for low-temperature bleaching)", Nature 369: 637-39 (1994).

19) Detergent compositions formulated as non-aqueous detergent liquids comprise a liquid nonionic surfactant (e.g., a linear alkoxylated primary alcohol), a builder system (e.g., a phosphate), an enzyme, and a base. The detergent may also contain anionic surfactants and/or a bleaching system.

The AmyDSM 90-related polypeptides can be incorporated into detergents at concentrations conventionally used in detergents. It is presently contemplated that in detergent compositions, the enzyme may be added in an amount corresponding to 0.00001-1.0mg (calculated as pure enzyme protein) AmyDSM 90-related polypeptide per liter of wash liquor.

In another embodiment, other enzymes, such as 2, 6- β -D-levan hydrolase, may be incorporated into detergent compositions comprising AmyDSM 90-related polypeptides and used to remove/clean biofilm present on household and/or industrial fabrics/clothing.

The detergent composition may for example be formulated as a manual (manual) or a machine (automatic) laundry detergent composition, including laundry additive compositions and rinse-added fabric softener compositions suitable for pre-treating stained fabrics, or as a detergent composition for general household hard surface cleaning operations or for manual or automatic dishwashing operations.

In a particular aspect, the detergent composition may comprise, in addition to the AmyDSM 90-related polypeptide, a 2, 6- β -D-levan hydrolase 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 starch lyase, a xylanase, an oxidase, a laccase, and/or a peroxidase and/or combinations thereof.

Generally, the properties of the enzyme selected should be compatible with the detergent selected (e.g., pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme should be present in an effective amount.

Protease: 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 protease (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 availableProteases include, but are not limited to:PRIMASE^TM、DURALASE^TM、and KANNASE^TM(Novo Nordisk A/S)；MAXACAL^TM、MAXAPEM^TM、PURAFECT OXP^TM、FN2^TMAnd FN3^TM(Genencor International，Inc.)。

Lipase: suitable lipases include those of bacterial or fungal origin. Including chemically modified, proteolytically modified or protein engineered mutants. Examples of useful lipases include, but are not limited to, lipases from the genus Humicola (Humicola) (synonym: thermophilic fungi (Thermomyces)), e.g., from Humicola lanuginosa (T. lanuginosus) (see, e.g., EP258068 and EP 305216) and H.insolens (see, e.g., WO 96/13580); pseudomonas lipases (e.g., from pseudomonas alcaligenes (p. alcaligenes) or pseudomonas pseudoalcaligenes (p. pseudoalcaligenes), see, e.g., european patent No. 218272), pseudomonas cepacia (p. cepacia) (see, e.g., european patent No. 331376), pseudomonas stutzeri (see, e.g., GB 1,372,034), pseudomonas fluorescens (p. fluoroscens), pseudomonas strain SD705 (see, e.g., WO 95/06720 and WO 96/27002), pseudomonas wisconsignis (p. wisconsinensis) (see, e.g., WO 96/12012); bacillus lipases (e.g., from Bacillus subtilis; see, e.g., Dartois et al, Biochemica et Biophysica Acta, 1131: 253-360(1993)), Bacillus stearothermophilus(see, for example, JP 64/744992) or Bacillus pumilus (see, for example, 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, WO95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, EP 407225 and EP 260105. Some commercially available lipases includeAnd LIPOLASE ULTRA^TM(Novo NordiskA/S)。

A polyesterase: suitable polyesterases, such as those described in, for example, WO 01/34899 and WO 01/14629, may be included in the compositions.

Amylase: the compositions may be combined with other amylases (e.g., non-production-enhancing alpha-amylases). These amylases include commercially available amylases, such as, but not limited toAnd BAN^TM(Novo Nordisk A/S)；And(from Genencor International, Inc.).

Cellulase: cellulase may be added to the composition. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from bacillus, pseudomonas, humicola, fusarium, rhizopus (Thielavia), Acremonium (Acremonium), e.g., U.S. Pat. nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757;and fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum (Fusarium oxysporum) as disclosed in WO 89/09259. Exemplary cellulases contemplated for use are those having color care benefits to fabrics. Examples of such cellulases are the cellulases described in e.g. EP 0495257, EP 0531372, WO 96/11262, WO 96/29397 and WO 98/08940. Other examples are cellulase variants, as described in WO 94/07998; WO 98/12307; WO 95/24471; PCT/DK 98/00299; EP 531315; U.S. Pat. nos. 5,457,046; 5,686,593; and those cellulase variants described in 5,763,254. Commercially available cellulases includeAnd(Novo NordiskA/S)；and(Genencor International, Inc.); and KAC-500(B)^TM(Kao Corporation)。

Peroxidase/oxidase: 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 (Coprinus), for example, Coprinus cinereus (C.cinereus) and variants thereof, such as those described in WO 93/24618, WO95/10602 and WO 98/15257. Commercially available peroxidases include, for example, GUARDZYME^TM(Novo Nordisk A/S)。

Detergent enzymes may be included in detergent compositions by adding individual additives containing one or more enzymes or by adding a combination additive comprising all of these enzymes. Detergent additives (i.e., individual additives or combination additives) may be formulated, for example, as granules, liquids, slurries, and the like. Exemplary detergent additive formulations include, but are not limited to, granules, particularly non-dusting granules, liquids, particularly stabilized liquids or slurries.

Non-dusting granules may be produced, for example, 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., polyethylene glycol, PEG) having an average molar weight of 1,000 to 20,000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which 15 to 80 ethylene oxide units are present; a fatty alcohol; a fatty acid; and fatty acids monoacylglycerols and diacylglycerols and triacylglycerols. Examples of film-forming coating materials suitable for application in fluid bed technology are given in, for example, GB 1483591. The liquid enzyme preparation may be stabilized according to established methods, for example by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid or boric acid. The protected enzymes may be prepared according to the methods disclosed in EP 238,216.

The detergent composition may be in any convenient form, for example, a bar, tablet, powder, granule, paste or liquid. Liquid detergents may be aqueous and typically contain 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 may optionally comprise one or more surfactants which may be nonionic (including semi-polar) and/or anionic and/or cationic and/or zwitterionic. The surfactant may be present in a wide range of about 0.1% to about 60% by weight.

When included in a detergent, the detergent will typically contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonate, alpha-olefin sulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxylated sulfate, secondary alkyl sulfonate, alpha-sulfo fatty acid methyl ester, alkyl or alkenyl succinic acid or soap.

When included in a detergent, the detergent will typically contain from about 0.2% to about 40% of a nonionic surfactant such as an alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or an N-acyl N-alkyl derivative of glucosamine ("glucamide").

The detergent may contain from 0 to about 65% of detergent builders or complexing agents, such as zeolites, diphosphates, triphosphates, phosphonates, carbonates, citrates, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl-or alkenylsuccinic acids, 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 such as polyacrylates, maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acid copolymers.

The enzymes of the detergent composition may be stabilized using conventional stabilizers, for example, a polyol (such as propylene glycol or glycerol), a sugar or sugar alcohol, lactic acid, boric acid or a boric acid derivative (such as an aromatic borate ester) or a phenyl boronic acid derivative (such as 4-formylphenylboronic acid). The composition may be formulated as described in WO 92/19709 and WO 92/19708.

It is presently contemplated that in detergent compositions, in particular, the enzyme variant may be added in an amount corresponding to from about 0.01 to about 100mg enzyme protein per liter of wash liquor (e.g., from about 0.05 to about 5.0mg enzyme protein per liter of wash liquor or from 0.1 to about 1.0mg enzyme protein per liter of wash liquor).

4.2Cleaning composition

In detergent applications, AmyDSM 90-related polypeptides are typically used in liquid compositions containing propylene glycol. The enzyme is dissolved, for example, in propylene glycol by mixing in a 25% v/v propylene glycol solution containing 10% calcium chloride.

The myDSM 90-related polypeptide discussed herein may be formulated in detergent compositions or other cleaning compositions for cleaning dishes. These compositions may be powders, gels or liquids. The composition may comprise the enzyme alone or together with other amylolytic enzymes and/or with other cleaning or bleach activating enzymes and other components common to cleaning compositions.

Accordingly, the dishwashing detergent composition may comprise a surfactant. The surfactant may be anionic, nonionic, cationic, amphoteric or a mixture of these types. The detergent may contain from 0% to about 90% by weight of a nonionic surfactant, such as a low to no foaming ethoxylated propoxylated straight chain alcohol.

The detergent composition may contain detergent builder salts of inorganic and/or organic type. Detergent builders can be subdivided into phosphorus-containing and non-phosphorus-containing types. Detergent compositions typically contain from about 1% to about 90% detergent builder. Examples of phosphorus-containing inorganic alkaline detergent builders, when present, include the water-soluble salts, especially the alkali metal pyrophosphates, orthophosphates and polyphosphates. When present, examples of phosphorus-containing organic alkaline detergent builders include the water-soluble phosphonates. Examples of inorganic phosphorus-free builders, when present, include water-soluble alkali metal carbonates, borates and silicates, as well as various types of water-insoluble crystalline or amorphous aluminosilicates of which zeolites are the best-known representative.

Examples of suitable organic builders include alkali metal, ammonium and substituted ammonium; a citrate salt; a succinate salt; a malonate salt; a fatty acid sulfonate; carboxymethoxysuccinate; ammonium polyacetate; a carboxylate; a polycarboxylate; an aminopolycarboxylate; polyacetylcarboxylates and polyhydroxysulfonates.

Other suitable organic builders include the high molecular weight polymers and copolymers known to have builder properties, for example, suitable polyacrylic acid, polymaleic acid and polyacrylic acid/polymaleic acid copolymers, and salts thereof.

The cleaning composition may contain bleaching agents of the chlorine/bromine type or of the oxygen type. Examples of inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite and hypobromite, and chlorinated trisodium phosphate. Examples of bleaching agents of the organic chlorine/bromine type are heterocyclic N-bromo and N-chloro imides, such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with water-soluble cations (e.g. potassium and sodium). Hydantoin compounds are also suitable.

The cleaning compositions may contain an oxygen bleach, for example in the form of inorganic perhydrochloride, optionally with a bleach precursor or as a peroxyacid compound. Common examples of suitable peroxyacid bleaching compounds are the tetrahydrate and monohydrate alkali metal perborates, alkali metal percarbonates, persilicates and perphosphates. Suitable activator materials include Tetraacetylethylenediamine (TAED) and triacetin. Enzymatic bleach activation systems may also be present in formulations such as perborate or percarbonate, triacetin and perhydrolases (see, e.g., WO 2005/056783).

The cleaning composition may be stabilized using conventional stabilizers for enzymes, for example, polyols (e.g., propylene glycol), sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters).

The cleaning compositions may also contain other conventional detergent ingredients such as deflocculants, fillers, suds suppressors, anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, dewatering agents, dyes, bactericides, fluorescers, thickeners and perfumes.

While the compositions and methods of this invention have been described with reference to the details below, it should be understood that various changes may be made.

4.3Method for evaluating amylase activity in detergent compositions

There are numerous alpha-amylase cleaning assays. Exemplary descriptions of testing for cleaning action include the following.

A "sample" is a piece of material, such as fabric, to which stain has been applied. The material may for example be a fabric made of cotton, polyester or a mixture of natural and synthetic fibres. The sample may also be paper, such as filter paper or nitrocellulose, or a piece of a hard material, such as ceramic, metal or glass. For amylases, stains are starch based, but may include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigments, oils or mixtures of these compounds.

A "smaller sample" is a piece of sample that has been cut with a single-well punch device or that has been cut with a custom-made 96-well punch, wherein the pattern of the multi-well punch matches that of a standard 96-well microtiter plate, or that has otherwise been removed from the sample. The sample may be fabric, paper, metal or other suitable material. Smaller samples may be stained before or after they are placed in the wells of a 24-well, 48-well, or 96-well microtiter plate. A "smaller sample" can also be made by applying a stain to a small piece of material. For example, a smaller sample may be a piece of stained fabric with a diameter of 5/8 "or 0.25". The custom punch is designed in such a way that it delivers 96 samples to all wells of a 96 well plate simultaneously. The device allows delivery of more than one sample per well by simply loading the same 96-well plate multiple times. Multi-well punch devices are contemplated to deliver samples simultaneously to any format of plate, including but not limited to 24-well, 48-well, and 96-well plates. In another contemplated method, the soiled test platform may be a bead made of metal, plastic, glass, ceramic, or other suitable material coated with a soil substrate. One or more of the coated beads are then placed into a 96-well, 48-well or 24-well plate or larger format plate containing a suitable bufferWash and enzyme wells. In this case, the released soil can be checked by direct absorbance measurements or on the supernatant after a secondary color reaction. Analysis of the released contaminants can also be performed by mass spectrometry. Yet another microscreen assay may be to deliver and fix a sample (e.g. indigo-dyed denim) to the wells of a multi-well plate, and add particles such as sand or larger particles, e.g. garnet sieved to include particles No. 6 to 8 or 9, and shake the plate, causing the sample to be rubbed by the added particles. This assay has been used for evaluation of cellulases in sand wash applications. The A can be measured by color release to the reaction buffer (e.g., the released indigo is dissolved in dimethyl sulfoxide and A is measured₆₀₀Absorbance at nm) or by reflectance measurements of abraded samples.

For example, when an untreated BMI (blood/milk/ink) sample is washed in a bleach-free detergent, most of the ink is released even without the assistance of a protease. The addition of protease leads to a small increase in ink release, which can be difficult to quantify over a large background. The compositions and methods of the present invention provide a treatment regimen that allows for control of the degree of stain fixation. As a result, samples may be produced that release variable amounts of stain, for example, when washed in the absence of the enzyme tested. The use of fixed samples resulted in a significant improvement in the signal to noise ratio in the wash assay. Furthermore, by varying the degree of immobilization, stains can be produced that give the best results under a variety of cleaning conditions.

Samples with known "strength" stains on various types of Materials are commercially available (EMPA, st. galen, switzerland; wfk-Testgewebe GmbH, kriffield, germany; or The dutch folbungen Center for Test Materials, Vlaardingen, The Netherlands) and/or can be made by practitioners (Morris and Prato, TextileResearch Journal 52 (4): 280-flac, (286 (1982)) other Test samples include, but are not limited to, blood/milk/ink (BMI) stains on cotton-containing fabrics, spinach stains on cotton-containing fabrics, or grass on cotton-containing fabrics, and chocolate/milk/soot on cotton-containing fabrics.

BMI stains 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 (Coomassie) stains fixed with 0.001% to 1% glutaraldehyde, or chocolate, milk and soot fixed with 0.001% to 1% glutaraldehyde.

The sample may also be agitated during incubation with the enzyme and/or detergent formulation. Wash performance data was dependent on the orientation of the sample in the wells (horizontal versus vertical), especially in 96-well plates. This would indicate that mixing during incubation was insufficient. Although there are numerous ways to ensure adequate shaking during incubation, a plate holder can be constructed in which the microtiter plate is sandwiched between two aluminum plates. This can be as simple as: for example, an adhesive plate cover is placed over the wells and the two aluminum plates are then clamped to a 96-well plate using any type of suitable commercially available clamp. The plate can then be placed on a commercial shaker. Setting the shaker to about 400 revolutions per minute produces very efficient mixing while the holder effectively prevents leakage or cross-contamination.

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 removed from the sample (see, e.g., Cayot and Taintuurier, anal. biochem. 249: 184-200, 1997). However, if the detergent or enzyme sample results in the formation of very small peptide fragments (e.g. due to the presence of peptidases in the sample), a larger TNBS signal, i.e. more "noise", is obtained.

Another means of measuring the cleaning performance of blood/milk/ink or other stains is based on ink release. Proteolysis of proteins on the sample results in the release of ink particles, which can be quantified by measuring wash liquor absorbance. The absorbance may be measured at any wavelength between 350nm and 800 nm. The absorbance was measured at 410nm or 620 nm. The wash liquor can also be tested to determine wash performance on stains containing grass, spinach, gelatin or coomassie stains. Exemplary wavelengths for these stains include 670nm for spinach or grass and 620nm for gelatin or coomassie. For example, an aliquot of the wash solution is removed (typically, for example, 100-150. mu.L from a 96-well microplate) and placed in a cuvette or multi-well microplate. It is then placed in a spectrophotometer and the absorbance is read at the appropriate wavelength.

The system may also be used to assay for enhanced enzymes and/or detergent compositions for dishwashing, for example using blood/milk/ink stains on a suitable substrate such as cloth, plastic or ceramic.

In one aspect, BMI stains are fixed to cotton by applying 0.3% hydrogen peroxide to a BMI/cotton sample for 30 minutes at 25 ℃, or 0.03% hydrogen peroxide to a BMI/cotton sample for 30 minutes at60 ℃. A smaller sample of approximately 0.25 "was cut from the BMI/cotton sample and placed in a well of a 96-well microtiter plate. A known mixture of detergent composition and enzyme (e.g., variant protein) is placed into each well. After an adhesive plate cover was placed on top of the microtiter plate, the microtiter plate was clamped to an aluminum plate and shaken on an orbital shaker at approximately 250 revolutions per minute for approximately 10 to 60 minutes. At the end of this time, the supernatant was transferred to the wells of a new microtiter plate and the absorbance of the ink was measured at 620 nm. This can similarly be used to test spinach or grass stains, which are fixed to cotton by applying 0.01% glutaraldehyde for 30 minutes at 25 ℃ to a spinach/cotton sample or grass/cotton sample. The same test can be performed with chocolate, milk and/or soot stains.

For ease of reading, the present document is organized into numerous paragraphs; however, the reader will understand that statements made in one paragraph may apply to other paragraphs. In this manner, headings used in various sections of this disclosure should not be construed as limiting.

To further illustrate the compositions and methods and their advantages, the following specific examples are given with the understanding that they are intended to be illustrative and not limiting.

Examples

Example 1

Cloning and isolation of the amyDSM90 Gene

The Bacillus megaterium strain DSM90 was obtained from Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSMZ), Inhoffentlass 7B, 38124 Brenrek, Germany. DNA was prepared and used for the isolation of the amyDSM90 alpha-amylase gene using a PCR primer set (SEQ ID NOs: 11 and 12) designed based on the Bacillus megaterium AAK00598 amylase sequence (SEQ ID NO: 3). Translation of the sequenced nucleotide of clone pME602 showed that the amylase from Bacillus megaterium DSM90(SEQ ID NO: 1) differs by 8 amino acids from the amylase from Bacillus megaterium AAK00598 (98.4% identity; FIG. 1).

Since this result was unexpected, the identity of bacillus megaterium strain DSM90 was confirmed by sequencing almost the entire 16S rRNA gene. The sequence results from the automatic base calling were confirmed manually to generate the final 1486nts sequence for analysis (SEQ ID NO: 8). Although the rRNA sequence of bacillus megaterium strain DSM90 appears to be unavailable in public databases, the sequence was determined using SEQ ID NO: 8 BLAST searches performed as queries returned a number of "hits" (99-100% identity) against the bacillus megaterium strain. Based on the available evidence, it is concluded that: the organism from which the subject DNA was obtained was a Bacillus megaterium strain. Although strain nomenclature is not important for the compositions and methods of the present invention, for the purposes of this disclosure, this strain will be referred to as DSM 90.

A search for similar amylase sequences was performed in public sequence databases using the BLASTp tool. Surprisingly, this search revealed that the most similar amylases (> 94% identity) were derived from Bacillus anthracis AAT32659(SEQ ID NO: 4), Bacillus thuringiensis AAT60457(SEQ ID NO: 5), Bacillus cereus AAP10417(SEQ ID NO: 6), and not from other Bacillus megaterium strains, with the only exception being the amylase from Bacillus megaterium AAK00598(SEQ ID NO: 3). The alignment of these orthologous amylase sequences is shown in figure 2 and their correlation (in terms of percent identity) is shown in (table 1).

TABLE 1 percent identity of orthologous amylase sequences

Phylogenetically, Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis form a tight cluster of highly related species and strains, with only long-range association with Bacillus megaterium (Ash et al, Letters in Applied Microbiology 13: 202-; 206, 1991). Thus, although it is expected that Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis contain similar amylases based on phylogenetic similarities, these amylases are surprisingly highly similar to Bacillus megaterium amylase. This indicates that AmyDSM90 is a member of the identifiable amylase subgroup, which also includes those amylases from Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. It is noteworthy that database annotations describe these bacillus cereus, bacillus anthracis, and bacillus thuringiensis amylases as "cytosolic," i.e., not exported or secreted outside the cell, an observation that is inconsistent with the findings of the present invention.

Example 2

Expression of AmyDSM90 in Bacillus licheniformis

For expression of the bacillus megaterium DSM90 amylase (AmyDSM90) in bacillus licheniformis, an intermediate construct was generated by using the pHPLT vector. pHPLT (FIG. 3; Solingen et al, extreme patents 5: 333-41, 2001) contains a thermostable amylase LAT promoter (pLAT) and a Bacillus licheniformis LAT signal peptide (pre LAT), followed by PstI and HpaI restriction sites for cloning.

The genomic DNA of Bacillus megaterium DSM90 was prepared from 1ml cell pellet of overnight incubated culture (tryptic Soy broth at 30 ℃) using the MASTERPURE gram-positive DNA purification kit from Epicentre according to the manufacturer's protocol. The amyDSM90 gene was amplified by PCR on a thermocycler (GE Healthcare) with PURETAQ READY-TO-GO PCR beads using the aforementioned primers based on the Bacillus megaterium AAK00598 amylase (SEQ ID NOs: 11 and 12), according TO the manufacturer's instructions (renaturation temperature 55 ℃). The resulting PCR fragment was digested with restriction enzymes PstI and HpaI and ligated into the digested pHPLTpDNA (50 ng/. mu.l, digested with restriction enzymes PstI and HpaI) with T4DNA ligase according to the supplier's instructions (Roche applied science, Indianapolis, IN, USA). The ligation mixture was transformed into Bacillus subtilis strain WW120 (also known as BG 3934; amyE:: xylRPxylAcomK-ermC). The transformation reaction was spread on LB agar plates containing 10ppm neomycin and 1% w/v insoluble starch. About 50-100 colonies were obtained.

Using colony PCR and sequencing, 20 colonies were evaluated for the presence of the insert. For colony PCR, each Bacillus subtilis transformant was suspended in 20. mu.L of sterile water, 2. mu.L of which was used in a PCR reaction containing READY-TO-GO TAQ PCR beads (Amersham), 22. mu.L of sterile water, 0.5. mu.L of 25. mu.M pHPLT-F1 primer (SEQ ID NO: 13), and 0.5. mu.L of 25. mu.M pHPLT-R1 primer (SEQ ID NO: 14). Cycling conditions were 94 ℃ for 4 minutes once, and a total of 30 cycles of 94 ℃ for 1 minute, 53 ℃ for 1 minute, and 72 ℃ for 2 minutes, followed by one final cycle at 72 ℃ for 7 minutes. mu.L of the PCR product was digested with Exo-SapIT (Amersham) to remove dNTPs and primers prior to sequencing. Sequencing was performed using primers pHPLT-SEQ-F1(SEQ ID NO: 15) and pHPLT-SEQ-R1(SEQ ID NO: 16) to confirm the sequence of the insert. The resulting plasmid was designated pME602.13 (also known as pHPLT-B. meg DSM90Amy, as on the plasmid map shown in FIG. 4). The cloned amylase gene has 8 amino acid changes compared to AAK00598 amylase (fig. 1). This plasmid can be used directly to express AmyDSM 90-related polypeptides, as described in example 3.

The pHPLT-B.meg DSM90Amy plasmid (DSM 90-wild type) was subsequently used to generate a Bacillus licheniformis AmyDSM90 expression strain in which AmyDSM90 was expressed as a fusion protein with the Bacillus licheniformis alpha-amylase (LAT) signal peptide (FIGS. 5 and 6, refer to SEQ ID NOs: 9 and 10). PCR amplification was performed using primers AmyPlatFW (SEQ ID NO: 17) and AmyTlat _ RV (SEQ ID NO: 18) and plasmid pHPLT-B.meg DSM90Amy as templates to generate a XhoI fragment containing the LAT-DSM90Amy precursor (i.e.an immature protein with a signal sequence) gene flanked at the 5 'end by the complete LAT promoter and at the 3' end by the LAT terminator. PCR was performed on a thermal cycler using PHUSION high fidelity DNA polymerase (Finnzymes OY, Espoo, Finland) according to the manufacturer's instructions (renaturation temperature 55 ℃). The resulting PCR fragment was digested with the restriction enzyme XhoI and ligated into XhoI-digested pICatH with T4DNA ligase according to the supplier's instructions (Invitrogen, Carlsbad, calif. The ligation mixture was transformed into bacillus subtilis strain SC6.1 as described in US patent application US20020182734 (international publication WO 02/14490). The sequence of The XhoI insert containing The LAT-AmyDSM90 precursor gene (BaseClear, Leiden, The Netherlands) was confirmed by DNA sequencing and The correct plasmid clone was designated pICatH-B.megDSM90amy (ori1) (FIG. 7).

The pICatH-B.meg DSM90amy (ori1) plasmid was then transformed into B.licheniformis strains BML612 and BML780 (derivatives of BRA7 and BML612, respectively; WO2005111203) at permissive temperature (37 ℃). Selecting one neomycin-resistant (neoR) and chloramphenicol-resistant (CmR) transformant from each transformation (designated BML612 (pICAtH-AmyDSM 90(ori1) and BML780 (pICAtH-AmyDSM 90(ori1), respectively.) by growing each transformed strain in a medium containing 5. mu.g/ml chloramphenicol at a non-permissive temperature (50 ℃), integrating these plasmids into the catH region of the B.licheniformis genome, selecting one chloramphenicol-resistant clone for each strain and growing it for several generations with chloramphenicol but no neomycin at a permissive temperature to loop out (loop-out) the vector sequence, and then one neomycin sensitive (neoS) CmR clone per strain was selected for further analysis, among these clones, the vector sequence for pICAtH (including the neomycin resistance gene) on the chromosome was excised and only the catH-LATAmyDSM90 cassette was left.

The catH-LATAmyDSM90 cassette on the chromosome was amplified by growing each strain in/on a medium with an increased chloramphenicol concentration. After several rounds of amplification, one clone of each strain resistant to 75. mu.g/ml chloramphenicol was selected and designated BML612-DSM90amyCAP75 and BML780-DSM90amyCAP75, respectively. To confirm the expression of AmyDSM90, the strains were incubated for 48 hours at 37 ℃ on heart infusion (Bacto) agar plates with 1% Starch Azure (Starch Azure) (potato Starch covalently linked to remazol brilliant blue R; Sigma-Aldrich, S7629). Clear bands indicating amylolytic activity were clearly visible around the colonies, indicating that a considerable amount of enzymatically active AmyDSM90 was expressed in the bacillus licheniformis strain.

Example 3

Expression of AmyDSM90 in Bacillus subtilis

Bacillus subtilis strain WW120 transformed with plasmid pme602.13 (described in example 2) secreted enzymatically active AmyDSM 90-related polypeptides as demonstrated by halo formation on starch plates after iodine staining. For the expression of AmyDSM 90-related polypeptides, cultures of this strain were typically stirred at 37 ℃ with 250 rpm in the following media (per liter): 10g Soytone, 75g glucose, 7.2g urea, 40mM MOPS, 4mM Tricine, 3mM dipotassium phosphate, 21.4mM KOH, 50mM NaCl, 276. mu.M potassium sulfate, 528. mu.M magnesium chloride, 50. mu.M trisodium citrate dihydrate, 100. mu.M calcium chloride dihydrate, 14. mu.M ferrous sulfate heptahydrate, 5.9. mu.M manganese sulfate dihydrate, 5.7. mu.M zinc sulfate monohydrate, 2.9. mu.M copper chloride dihydrate, 4.2. mu.M cobalt hexahydrate, and 4.5. mu.M sodium molybdate dihydrate were incubated for 60 to 72 hours. For a volume of 1L, all components (except soytone) were mixed at 500mL, sterile filtered and added to an aliquot of 2X soytone that had been sterilized by autoclaving. Trace metals and citrate were made into 100X or 1000X mother liquor. The buffer, potassium hydroxide, sodium chloride, potassium sulfate, magnesium chloride and trace metals were made into 10X mother liquor. After mixing all the components, the pH was adjusted to 7.3. Before use, the medium was supplemented with 20mM calcium chloride and 10mg/L neomycin trithionate. All experiments described herein used AmyDSM90 expressed in bacillus subtilis.

Example 4

Purification of AmyDSM90

Growth medium from the shake flask described in example 3 (500mL) including the expressed AmyDSM90 was concentrated to a volume of 25 mL. 25ml of 50mM MES buffer, pH 5.8, containing 2mM calcium chloride was then added to the concentrate followed by 6.6g ammonium sulfate to give a final concentration of 1M ammonium sulfate. After filtration, the sample was applied to a column using 5mL phenyl sepharose (phenylsepharose) equilibrated with a buffer containing 50mM MES, pH 5.8, 2mM calcium chloride and 1M ammonium sulfate. After loading, the column was washed with 8 volumes of the same buffer as used for equilibration. AmyDMS 90-related polypeptide was eluted from the column over 12 column volumes using a gradient starting with equilibration/wash buffer and ending with 50mM MES, pH 5.8, 2mM calcium chloride buffer (no ammonium sulfate). The conditions of 50mM MES, pH 5.8, 2mM calcium chloride buffer (no ammonium sulfate) were extended for 7 column volumes and then 40% propylene glycol was included in the buffer for 5 column volumes. The column was washed with water for 5 column volumes in a countercurrent direction and 10 column volumes in a cocurrent direction. Amylase began eluting 9 column volumes into the gradient and continued eluting over 2 column volumes.

Alpha-amylase activity was measured using the Megazyme amylase activity assay, and all active fractions were pooled, concentrated (using a 5K VIVASPIN 20mL centrifugal concentrator) and buffer exchanged three times to 50mM MES, pH 5.8, 2mM calcium chloride buffer to remove residual ammonium sulfate.

Example 5

Expression of AmyDSM 90-related polypeptides in Bacillus licheniformis

Variants of AmyDSM90 were generated using the QUICK CHANGE multi-site mutagenesis method (Stratagene, La Jolla, Calif.) [ e.g., (a) deletion of R179-G180 (. DELTA.RG), (b) substitution of M200L and (c) a combination of these mutations ].

Each variant (i.e., comprising R179 and G180 deletions and M200L substitutions, respectively) was first generated using the QUIK-CHANGE multi-site mutagenesis kit along with the corresponding primer pairs RGF (SEQ ID NO: 21)/RGR (SEQ ID NO: 22) and M200F (SEQ ID NO: 19)/M200R (SEQ ID NO: 20). The Δ RG variant serves as a template for a second QUIK-CHANGE multi-site mutagenesis reaction using primer pair M200F/M200R, aimed at combining RG deletions with M200L substitutions. All 3 variants were generated using the protocol outlined below:

pHPLT-B.meg DSM90Amy was methylated using dam methylase (NEB, Massachusetts, USA) prior to Quik-CHANGE mutagenesis. In addition to using complimentary primers instead of a single forward primer in the reaction, a QUIK-CHANGE multi-site mutagenesis kit was used, according to which pHPLT-B.meg DSM90Amy served as the template to generate the first two variants. Using the GE Healthcare Illustra Templiphi kit (GE Healthcare), 1. mu.L of QUICK-CHANGE reactant served as template for rolling circle amplification according to the kit protocol. The rolling circle reaction was diluted 10-fold and 1. mu.L was transformed into 100. mu.L competent Bacillus subtilis BG6006 cells (degUHy32, oppA, dspoII3501, amyE:: xylRPxylAcomK-ermC, daprE, dnprE, depr, dispA, dbpr, dvpr, dwwrA, dmpr-ybfJ, dnprB) (US20050202535A 1). The transformation was spread on LB agar plates containing 10ppm neomycin and 1% w/v insoluble starch. About 100 colonies were obtained. Using colony PCR and sequencing, 4 colonies were evaluated for the presence of the desired mutation. For colony PCR, each Bacillus subtilis transformant was suspended in 20. mu.L of sterile water, 2. mu.L of which was used in a PCR reaction containing READY-TO-GO TAQ PCR beads from Amersham, 22. mu.L of sterile water, 0.5. mu.L of 25. mu.M pHPLT-F1 primer (SEQ ID NO: 13) (5'-TACATATGAGTTATGCAGTTTG-3') and 0.5. mu.L of 25. mu.M pHPLT-R1 primer (SEQ ID NO: 14) (5'-GTTATGAGTTAGTTCAAATTCG-3'). Cycling conditions were once at 94 ℃ for 4 minutes; a total of 30 cycles of 94 ℃ for 1 minute, 53 ℃ for 1 minute, 72 ℃ for 2 minutes, followed by one final cycle at 72 ℃ for 7 minutes. mu.L of the PCR product was digested with Exo-SapIT from Amersham to remove dNTPs and primers prior to sequencing. Sequencing was performed using primers pHPLT-SEQ-F1(SEQ ID NO: 15) and pHPLT-SEQ-R1(SEQ ID NO: 16) to confirm the presence of the desired mutation.

The pHPLT-b.meg DSM90Amy plasmid (i.e., with R179-G180 deletion, M200L substitution, or both) served as a template to construct a bacillus licheniformis AmyDSM90 expression strain as described in example 2.

Example 6

Gene expression of AmyDSM 90-related polypeptides using codon modification

Synthetic, codon-optimized DNA fragments encoding each of the 4 AmyDSM 90-related polypeptides (i.e., naturally occurring orthologs) were generated by GeneArt GmbH (Regensburg, Germany) and used to construct additional bacillus subtilis strains (table 2).

TABLE 2 synthetic DNA fragments containing modified codons

The pHPLT vector (FIG. 3; example 2) was used for expression of these AmyDSM 90-related polypeptides. The synthesized genes (i.e., SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25 and SEQ ID NO 26) were digested with restriction enzymes PstI and HpaI, and ligated into pHPLT pDNA (50 ng/. mu.l) digested with restriction enzymes PstI and HpaI with T4DNA ligase according to the supplier's instructions (Roche applied Science, Indianapolis, IN, USA). The ligation mixture was transformed into Bacillus subtilis strain WW120 and subjected to sequence analysis as described in example 2.

To confirm the expression of the AmyDSM 90-related polypeptide, each transformed bacterium was incubated for 24 hours at 37 ℃ on heart infusion (Bacto) agar plates with 10mg/l neomycin and 1% starch sky blue. Clear zones indicating amylolytic activity were clearly visible around all colonies. These results show that a substantial amount of AmyDSM 90-related polypeptides are expressed in bacillus subtilis and have amylase activity.

Example 7

Megazyme assay for alpha-amylase activity

The Megazyme assay for measuring alpha-amylase activity was performed as follows:

materials:

25mM Bis Tris propane buffer pH 6.9 and 2mM CaCl₂，

BPNPG7 substrate, 5.45mg/mL (Amylase HR Reagent, (blocked) p-nitrophenyl a-D-maltoheptoside, Cat. No.: R-AMHR4Megazyme International),

amyloglucosidase, 10U/mL, and

alpha-glucosidase, 10U/mL.

The method comprises the following steps:

1. the substrate was prepared by adding 10mL of distilled water to a vial containing BPNPG7 along with amyloglucosidase/alpha-glucosidase. A1 mL aliquot was prepared and stored at-20 ℃.

2. The hot mixer was set to 43 ℃.

3. Adjust plate reader to the following settings: 40 ℃; 405nm, kinetics, 45 min, 15 sec interval, premix 7 sec.

4. With BTP pH 6.9 and 2mM CaCl₂Buffer preparation enzyme was diluted to 1ppm and stored on ice.

5. Add 10. mu.L of substrate to 150. mu.L of BTP pH 6.9 and 2mM CaCl₂And (4) a buffer solution. The substrate solution was prepared in large volumes and 160 μ Ι _ of solution was dispensed to each microtiter plate well of the plate pre-warmed to 40 ℃ for at least 10 minutes.

6. While the plate was left on the warm platform, 10 μ L of enzyme was added to each well.

7. The plates were immediately transferred to a plate reader and the rate of enzyme reaction was monitored for 45 minutes, or until sufficient linear data was obtained.

8. The specific activity was calculated by dividing the rate obtained (milliOD/min) by μ g of enzyme used in the assay.

Example 8

Performance of AmyDSM 90-related polypeptides in cleaning action screening assays

Partially purified AmyDSM 90-related polypeptides were analyzed in a 96-well CS 28-orange-stained rice starch soiled fabric swatch micro-application cleaning assay (example 4). 1/4 inch discs were cut from CS28 rice starch stained fabric (Test Fabrics catalog number CS-28; Test Fabrics Inc) using a fabric punch and two discs were placed in each well of a flat bottom 96 well Test plate. Prior to performing the amylase assay, the assay plate was placed in a thermal mixer and incubated at 25 ℃ with shaking at 750 rpm for 1 hour by adding 200 μ L of water to each well, and the samples were pre-washed to remove any dye that was not tightly bound. The wash solution was removed from each well and the sample discs were rinsed once in 200 μ L of water. The rinse water was immediately discarded and the washed sample discs were air dried in the test plates at 37 ℃.

For the alpha-amylase cleaning assay, a preselected buffer is added to each well and equilibrated to a preselected temperature. In the experiments of the invention, the assays were carried out in 25mM BTP (pH 8.0) or in 25mM CPPS (pH 10.3) with each buffer solution additionally containing 2mM CaCl₂. 200 μ L of pre-selected buffer was added to each well and allowed to equilibrate to the desired temperature (i.e., 20 ℃ or 40 ℃) for 15 minutes. Add 10. mu.L of enzyme solution to each well and incubate the plates at the same temperature with shaking at 750 rpm for 1 hour. After transfer of 150 μ L of assay solution to fresh microtiter plates, enzyme performance was established by the amount of enzyme-dependent color release quantified spectrophotometrically at 488 nm. For additional information on this assay, see U.S. patent No. 7,122,334.

The results are shown in FIG. 8-Shown in the graph in fig. 11. AmyDSM90 was highly effective at removing starchy stains from fabric samples at both pH 8 and pH 10.3 at 40 ℃, while it was more effective than the control enzyme (i.e.,genencor international, palo Alto, CA, USA). Surprisingly, the enzyme was also significantly effective at lower temperatures, i.e. 20 ℃ at pH 8 and pH 10.3, while it was more effective than the control enzymeAnd(both from Genencor International, Palo Alto, Calif., USA) showed better stain removal.

The 96-well format of the assay is suitable for high throughput screening. A larger sample is used whose reflectance can be measured and the 24-well plate form of the assay is used to confirm the results. Both measures (i.e., supernatant absorbance and sample reflectance) show near perfect correlation (coefficient of determination) r²Has a value of 0.99), thus confirming the result.

Example 9

Wash performance of AmyDSM 90-related polypeptides in detergents

The wash performance Test was performed using a sample of CS28 rice starch stained fabric indicating that the dye was combined with starch (Test Fabrics catalog number CS-28; Test Fabrics Inc.). Prior to the assay, the dyed test fabrics were pre-washed with distilled water for 1 hour and air dried. Pre-washing of the test fabrics removed any poorly bound indicator dye prior to the assay. Alternatively, the samples are pre-washed after they have been loaded into the wells of the microtiter plate (as described in example 8).

1/4 inch discs were cut from the pre-washed fabric and placed in 96 well plates. Add 190 μ L of detergent solution to each well (e.g.,IEC-A^＊base detergents (Cat. No. 88010-1; WFKTestgeweebe GmbH, Germany). The plates with the samples and detergent were preincubated for 15 minutes at 40 ℃. Purified AmyDSM 90-related polypeptide from example 4 or 0 to 1ppm of a commercial alpha-amylase (e.g.,) Or(as a control) the amylase assay was started. Wash performance was determined by measuring the color release of the indicator dye into the solution phase as measured by spectrophotometry at 488 nm.

Example 10

Multiple AmyDSM 90-related polypeptides in a cleaning action screening assay

Naturally occurring variants of AmyDSM90, i.e., bacillus anthracis AAT32659 amylase, bacillus cereus AAP10417 amylase, bacillus thuringiensis AAT60457 and bacillus megaterium AAK00598 amylase, were expressed in bacillus subtilis BG6006 and tested for their cleaning efficiency essentially as described in example 8. The assay was performed in 25mM HEPES (pH 8.0) or 25mM CAPS (pH 10.3) buffers, each buffer having a water hardness of 150ppm Ca²⁺And Mg²⁺(2: 1 Ca: Mg). 209 μ L of buffer was added to each well of a 96-well microtiter plate and allowed to equilibrate to the desired temperature (i.e., 40 ℃) for 15 minutes. Add 11. mu.L of purified enzyme solution to each well and incubate the plates at the same temperature with shaking at 750 rpm for 1 hour. As previously described, enzyme performance was based on the amount of enzyme-dependent color release, which was quantified spectrophotometrically at 488nm after transfer of 150. mu.L of reaction solution to a fresh microtiter plate.

The results shown in FIGS. 12 and 13 show similar cleaning ability (i.e., starch removal) of orthologous amylases compared to AmyDSM90 and Amy707(Tsukamoto et al, biochem. Biophys. Res. Comm.151: 25-31, 1988).

Example 11

Terg-o-meter test of AmyDSM 90-related polypeptides

The Terg-o-meter provides an intermediate laboratory scale test assay for detergents and enzymes that mimics the top loading, paddle action (paddle action) type washing machine common in north america. The conditions and procedures used to perform the assay are described below:

sample preparation:

EMPA161 dyed corn starch (EMPA Testmaterial AG, St. Gallen, Switzerland) and on cotton

CS-28 dyed Rice starch on Cotton (Test fabrics, Inc., Center for testing materials, Folar Ding Netherlands)

A detergent:

AATCC liquid, pH 7(1.5g/L) and

AATCC powder, pH10.

Amylase:

bacillus megaterium DSM90(AmyDSM90),

bacillus megaterium AAK00598, and

OxAmas a control.

Conditions are as follows:

1 liter of a Terg-o-meter,

4 parts starch stained sample plus 4 parts white bleached cotton sample for ballasting

6gPg (about 100ppm [ 3: 1, Ca: Mg ]) water hardness, and

washing temperature of 23 ℃.

The method comprises the following steps:

the center of the soil sample was pre-read twice on a black background using a Minolta reflectometer. Add 1L of deionized water to the tert-o-meter followed by pre-selected detergent dosage and salt to create pre-selected water hardness. The solution was mixed for 5 minutes and allowed to reach 74 ° F wash temperature. The pH of the terg-o-meter was measured and the test amylase was added. 4 parts of the starch stained sample and 4 parts of the white bleached cotton sample were added and the terg-o-meter was started. The sample was mixed in the detergent solution at 100 rpm for 15 minutes. After incubation, the samples were transferred to a 4L plastic beaker, rinsed for 3 minutes under running tap water and running deionized water (from below) and placed in a front-end washing machine for the spin cycle (spin cycle). Multiple samples were dried overnight by spreading on a clean sheet. The center of the soil on the sample was read twice on top of the black background. The results are reported as soil removal index (% Δ SRI).

As a result:

the results are shown in FIGS. 14 to 16 and compared to OXAMIn contrast, the amylase of the invention exhibits excellent laundry performance in liquid and powder detergents.

Example 12

Launder-o-meter test of AmyDSM 90-related polypeptides

Launder-o-meter (lom) provides an intermediate laboratory scale test assay for detergents and enzymes that mimics the front-end, drum washing machine common in europe. The conditions and procedures used to perform the assay are described below:

sample preparation:

EMPA161 dyed corn starch (EMPA Testmaterial AG, St. Gallen, Switzerland) and on cotton

CS-28 dyed Rice starch on Cotton (Test fabrics, Inc., Center for testing materials, Folar Ding Netherlands)

A detergent:

IEC A standard detergent, pH10.7(6.16g/L),

IEC A with bleaching agent, pH 10.4

Commercially available gel laundry detergents.

Amylase:

bacillus megaterium DSM90(AmyDSM90), wild type

Bacillus megaterium DSM90 mutant M200L,

bacillus megaterium DSM90 mutant DELTA RG,

bacillus megaterium DSM90 mutants M200L and Δ RG,

OXAMas a control, and

bacillus licheniformis amylase (LAT, Genencor International) was used as a control.

Conditions are as follows:

each test had 4 samples of starch contamination,

12gPg (about 200ppm [ 3: 1, Ca: Mg ]), water hardness, and

washing temperature of 40 ℃.

The method comprises the following steps:

the LOM is set to the appropriate temperature. The wash cycle was started at room temperature and the LOM was allowed to climb to the desired temperature in a 45 minute wash cycle. The center of the soil sample was pre-read twice on a black background using a 50mm Minolta reflectometer. 200mL of the wash solution was added. A stainless steel ball (6) and test enzyme sample are added and mixed thoroughly, followed by the addition of the washed sample. The test beaker was covered and clamped and placed in the LOM. After 45 minutes incubation, the sample was removed from the test beaker, excess water was squeezed out and transferred to a 4L plastic beaker. The samples were rinsed under running tap water for 3 minutes, placed in a front-end washing machine for the spin cycle (spin cycle) and spread out to air dry overnight. The center of the soil on the sample was read twice on top of the black background. The results are reported as soil removal index (% Δ SRI). The results are shown in fig. 17 to 21. Compared to commercial enzymes (OXAM and LAT), AmyDSM90 has improved wash performance in both standard and commercial detergents, e.g., variant M200L produced performance benefits in bleach-containing detergents (fig. 17 and 18).

All references mentioned herein are incorporated by reference in their entirety.

Claims (34)

1. An isolated polypeptide having at least 80% amino acid sequence identity to AmyDSM90(SEQ ID NO: 1), wherein the polypeptide has at least one amino acid sequence identity to SEQ ID NO: 1 has at least one of the following features:

a) an aspartic acid at position 21 of a compound of formula (I),

b) asparagine at position 97, and

c) isoleucine at position 128.

2. The polypeptide of claim 1, having an aspartic acid at position 21 and an asparagine at position 97.

3. The polypeptide of claim 1, having an aspartic acid at position 21 and an isoleucine at position 128.

4. The polypeptide of claim 1, having an asparagine at position 97 and an isoleucine at position 128.

5. The polypeptide of claim 1, having an aspartic acid at position 21, an asparagine at position 97, and an isoleucine at position 128.

6. The polypeptide of claim 1, which is expressed as a secreted polypeptide by a heterologous cell.

7. The polypeptide of claim 1, which has alpha-amylase activity.

8. The polypeptide of claim 1, which hybridizes to SEQ ID NO: 1 has at least 90% identity.

9. The polypeptide of claim 1, which hybridizes to SEQ ID NO: 1 has at least 95% identity.

10. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 1.

11. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

12. An isolated polypeptide having the amino acid sequence of SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25 or SEQ ID NO: 26.

13. A composition comprising the polypeptide of any one of claims 1-12.

14. The composition of claim 13, wherein the composition is a cleaning composition.

15. The composition of claim 13, wherein the composition is effective for removing starchy stains from laundry.

16. The composition of claim 13, wherein the composition is effective for removing starchy stains from dishware.

17. The composition of claim 13, wherein the composition is effective for removing starchy stains from fabrics.

18. A composition comprising a polypeptide having the sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

19. The composition of claim 18, wherein the composition is a cleaning composition.

20. The composition of claim 18, wherein the composition is effective for removing starchy stains from laundry.

21. The composition of claim 18, wherein the composition is effective for removing starchy stains from dishware.

22. The composition of claim 18, wherein the composition is effective for removing starchy stains from fabrics.

23. A method for removing starchy stains from a surface comprising

Incubating the surface in the presence of an aqueous composition comprising an effective amount of an alpha-amylase having at least 80% amino acid sequence identity to AmyDSM90(SEQ ID NO: 1), wherein the polypeptide has an amino acid sequence as compared to the amino acid sequence of SEQ ID NO: 1 has at least one of the following features:

a) an aspartic acid at position 21 of a compound of formula (I),

b) asparagine at position 97, and

c) isoleucine at position 128;

allowing the alpha-amylase to hydrolyze a starch component present in the starchy stain to produce starch-derived smaller molecules that are solubilized in the aqueous composition,

thereby removing starchy stains from the surface.

24. The method of claim 23, wherein the alpha-amylase is AmyDSM90(SEQ ID NO: 1).

25. The method of claim 23, wherein the alpha-amylase is a variant AmyDSM90(SEQ ID NO: 28) having a deletion of residues R179 and G180.

26. The method of claim 23, wherein the alpha-amylase is a AmyDSM90 variant (SEQ ID NO: 29) having a substitution at M200.

27. The method of claim 23, wherein the alpha-amylase is a AmyDSM90 variant (SEQ ID NO: 30) having a deletion of residues R179 and G180 and a substitution at M200.

28. A method for removing starchy stains from a surface comprising

Incubating the surface in the presence of an aqueous composition comprising an effective amount of a peptide having SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID

NO: 6. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 29 or SEQ ID NO: 30.

29. The method of any one of claims 23-28, wherein the surface is a fabric surface.

30. The method of any one of claims 23-28, wherein the surface is on a dish.

31. The method of any one of claims 23-28, wherein the surface is a garment surface.

32. A method for expressing an alpha-amylase, comprising:

introducing into a host cell an expression vector comprising a polynucleotide encoding an alpha-amylase having at least 80% amino acid sequence identity to AmyDSM90(SEQ ID NO: 1), wherein the polynucleotide is fused in-frame to a signal sequence;

expressing the alpha-amylase as a secreted polypeptide into the host cell culture medium; and

recovering the secreted alpha-amylase from the host cell growth medium;

thus isolating the alpha-amylase as a secreted polypeptide.

33. The method of claim 32, wherein the signal sequence is a native signal sequence.

34. The method of claim 32, wherein the signal sequence is from Bacillus (Bacillus) AmyE or AprE or Streptomyces (Streptomyces) CelA.